Experiment 1 Restriction Digestion for testing · Benchling

Experiment 1 Restriction Digestion for testing

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Wangui Mbuguiro

Dates: 2016-04-18 to 2016-05-07

Monday, 4/18/16

Goal:

Enzymes that work:

	A	B	C	D	E
1		Tal14:meYFP		TRE:TALER14	phef1a:rtTA
2		Tal14	mEYFP
3		sacI	BlpI **BAD ENZYME	StuI	BsrGI
4		StuI		BsrGI	BspMI
5		Xho			ApaI
6					SacI
7
8	Final Choice:	sacI	BlpI	StuI	BspMI

Table1

	A	B
1	Tal14:mEYFP 1	no enzyme
2	Tal14:mEYFP 1	BlpI
3	Tal14:mEYFP 1	sacI
4	Tal14:mEYFP 1	BlpI, sacI
5	Tal4:mEYFP 2	BlpI, SacI
6	Tal4:mEYFP 3	BlpI, SacI
7	Tal4:mEYFP 4	BlpI, SacI
8	TRE: TALER14 1	no enzyme
9	TRE: TALER14 2	StuI
10	TRE: TALER14 3	StuI
11	TRE: TALER14 4	StuI
12	TRE: TALER14 1	StuI
13	hef1a: rtTa 1	no enzyme
14	hef1a: rtTa 1	BspMI
15	hef1a: rtTa 2	BspMI
16	hef1a: rtTa 3	BspMI
17	hef1a: rtTa 4	BspMI

Table2

Notes for digest:

	A	B	C
1	plasmid	buffer	temperature
2	Tal14:eYFP	cutsmart	37
3	TRE:Tal14	cutsmart	37
4	hef1a:rTTA	buffer 3.1	37

Table3

digestcalculations.jpg

Pouring an Agarose Gel: 10uL of SYBRSafe added to agarose solution after micorwaving and allowing it to cool until touchable

Results:

clipboard_2016-04-30_15:09:09.png

clipboard_2016-04-30_15:09:34.png

weisslab 2016-05-08 17hr 33min (1).jpg

Saturday, 5/7/16

Enzyme choices:

●

Tal14:mEYFP1: AseI, BspmI

●

TRE: TALER14: StuI

●

hef1a:rtTa:apaI

	A	B	C	D
1	Lane	Sample	Enzyme	Expected?
2	1	Ladder	--	--
3	2	pDEST control	AseI BspmI	Yes
4	3	pDEST control	AseI	Yes
5	4	pDEST control	BspmI	kinda
6	5	Tal14:mEYFP 1a control	no enzyme	Yes
7	6	Tal14:mEYFP 1a	AseI	Yes
8	7	Tal14:mEYFP 1a	BspmI	Yes
9	8	Tal14:mEYFP 1a	AseI BspmI	Yes
10	9	Tal14:mEYFP 1b	AseI	Yes
11	10	Tal14:mEYFP 1b	BspmI	No
12	11	Tal14:mEYFP 1b	AseI BspmI	Yes
13	12	blank	--	--
14	13	TRE: TALER14 1a control	no enzyme	Yes
15	14	TRE: TALER14 1a	StuI	Yes
16	15	TRE: TALER14 1b	StuI	Yes
17	16	pDEST control	ApaI	Yes
18	17	hef1a: rtTa 1a control	no enzyme	Yes
19	18	hef1a: rtTa 1a	ApaI	Yes
20	19	hef1a: rtTa 1b	ApaI	Yes

Table4

MINIPREPS CHOSEN:

Tal14:mEYFP: 1a

TRE:Tal14: 1a

hef1a:rtTA: 1a

weisslab 2016-05-08 17hr 33min.jpg

Image with both side by side:

clipboard_2016-06-30_11:05:28.png

Notes for digest:

	A	B	C
1	plasmid	buffer	temperature
2	Tal14:eYFP	3.1	37
3	TRE:Tal14	cutsmart	37
4	hef1a:rTTA	cutsmart	25

Table5

Diagnostic Restriction Digestion (Maya, Liz, Wangui, Kathryn 4/18/16)

Introduction

A diagnostic restriction digest helps identify correctly assembled clones from incorrect clones.

Materials

200 ul PCR strip tubes, one per reaction
Restriction enzyme (chosen below), 1 ul per reaction.
10X restriction enzyme buffer (chosen below), 1 ul per reaction.

Procedure

Choose A Good Restriction Enzyme

Using Benchling, choose a restriction enzyme that meets the following criteria:

- Cuts at least once in the insert (for a pEXPR, either the promoter or the gene.)

- Cuts at least once in the backbone.

- Gives bands that aren't too large (> 8 kb) or too small (< 200 bp).

- Gives a band pattern that is significantly different from the expected error mode.

* For LR reactions, the most common error is a pDEST that slipped through the selection.

- Begin in the "Brian's Favorites" list, then expand to the main Weiss lab list.

- If you can't find a single enzyme that gives an acceptable band pattern, choose two enzymes that give a acceptable band pattern when used together.

* This double-digest is subject to buffer compatibility, outlined below.

Record your enzyme choice on the plasmid's Description page.

Benchling will tell you the enzyme's buffer compatibility and active temperature. Record the buffer in which the enzyme is most active.

If there are multiple bufers in which the enzyme is equally active, choose in this order: Buffer 3.1, Buffer CS, Buffer 2.1, Buffer 1.1.

Set up the restriction digest

Retrieve the minipreps and the appropriate 10X buffer concentrate from the freezer. Thaw on the benchtop or in your fingers.

Label the PCR tubes with your initials and an incrementing number.

ie: BT-1, -2, -3, -4

Vortex the minipreps and the 10X buffer concentrate briefly, then pulse down in the microfuge.

For each miniprep, set up a PCR tube containing the following in order:

- 5 ul enzyme-quality H2O

- 1 ul enzyme buffer

- 3 ul miniprep DNA

- 1 ul enzyme

Remove the enzyme from the freezer for as little time as possible.

I have specified an "arbitrary" 3 µl volume of miniprep DNA; this should be fine as long as your miniprep concentration is >= 100 ng/ul.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Incubate at the appropriate temperature for at least 1 hour and more more than 16 hours.

If the enzyme's active temperature is 37°C, use the 37°C plate incubator.

Stop the reaction by adding 2 ul of 6X NEB purple gel loading dye to each reaction.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

PAUSE POINT: The reaction can be stored almost indefinitely at room temperature once it's been stopped.

Proceed to gel electrophoresis.

Pouring an agarose gel

Introduction

Agarose gels are used to separate DNA fragments. Their most common use is

Materials

TAE buffer, 50-100 ml
- There is generally 1X TAE buffer at the iGEM bench in a 1L or 2L bottle.
- If not, mix some more up from the carboy 10X stock by the main lab gel station. (Use the 1L graduated cylinder at the gel station.)
UltraPure agarose
Casting tray, casting stand, combs

Procedure

Mix and melt the agarose

Check to see if there is a gel waiting in the fridge.

Determine how much molten agarose you'll need.

A small gel cast takes 50 ml; a large gel cast is 100 ml.

Measure out the appropriate amount of TAE into a glass bottle or flask.

Add 5 µl SYBRSafe (0.5X ; stock solution is 10000X) per 100 ml of the solution and mix well.

Add UltraPure agarose to a final concentration of 1% (mass / volume)

So, if you're making 50 ml gel: 0.5 g.

Swirl the bottle or flask to distribute the agarose.

Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. ~30 seconds/100ml solution

Let sit until cool enough to handle.

The agarose MUST cool some -- if it's too hot, it can warp the casting trays.

Don't allow it to get too cold!

PAUSE POINT - You can put the flask in the 55°C water bath almost indefinitely at this point.

Pour the gel

Set up the gel tray in the casting stand. Make sure the rubber gaskets are flat up against the edges of the casting tray.

Set up the gel combs to form the wells.

Rinse the combs with water and wipe dry.

Note for combs: 15-well combs hold about 6 ul liquid per well, 12-well combs hold about 15 ul per well, 8-well combs hold about 20 ul per well

Taping two 8-well comb wells together results in a well that holds up to 100 ul

Taping three 8-well comb wells together result in a well that holds up to 200 ul

Pour the molten agarose into the casting tray.

If bubbles form around the combs, remove and re-insert.

Wait 30 mintues for the gel to solidify.

Use immediately, or place in a plastic zip-lock baggie with a little 1X TAE and store at 4°C.

Gel electrophoresis

Introduction

Gel electrophoresis separates pieces of DNA by length.

Materials

Agarose gel, 1 lane per sample, plus at least 1 lane for the ladder.
- If you just poured the gel, make sure it has had at least 30 minutes to set.
- If you have a large gel but only need a few lanes, cut out just a piece of it with a razor blade. Cut straight!
Samples to run
6X NEB Purple Loading Dye
Molecular weight standard (aka "ladder")
- Commonly available ladders are Hyperladder I and NEB 2-Log.
Gel box, lid, leads
Electrophoresis power supply
1X TAE buffer, enough to fill the gel box.

Procedure

Prepare your samples

If your samples are frozen, thaw them completely, flick or vortex to mix, then pulse down in the microfuge.

If your samples are not already in loading dye, mix them 1:6 with 6X NEB Purple Loading Dye

Add 1 µl of loading dye for every 5 µl of sample.

Pro tip: If you don't want to mix loading gel with your entire sample (PCR products, for example), cut off a strip of Parafilm; pipette 2-3 ul of sample onto the parafilm; add 1 ul loading dye; mix by pipetting and load directly.

Set up the gel box

Program the voltage on the power supply. For the small gel box, use 100V; for the large gel box, use 150V.

Program the time on the power supply. For a small analytical gel, set the timer for 30 minutes.

For larger gels, start at 45 minutes, and then check regularly!

Attach the leads to the gel box cover. Make sure that the red (positive) lead is attached to the side of the box farthest away from the wells in the gel.

Remember, the DNA is negatively charged and will move toward the positive terminal. A useful neumonic for remembering this is "Run to Red."

If the gel box has not been used previously that day, empty it and rinse it out with DI water.

The TAE buffer can be re-used, but if it's been sitting out for too long it evaporates and the salt concentration (and conductivity) changes. TAE is cheap; when in doubt, replace it.

Place the gel in the gel box.

Pour TAE into the gel box until it just barely covers the gel.

The TAE's purpose is to conduct electricity; over-filling the gel box results in a larger conductive path, more current flow, and more heating (which can screw up your run.)

Check the wells to see that they are free of bubbles. If there are bubbles, blow them out by pipetting 100 µl of TAE from the gel box into the well.

Load your samples

Load your samples. For large combs, load 10 µl; for small combs, load 5 µl.

Work quickly. The samples begin to diffuse in the buffer, leading to smeared bands.

Load the ladder in the last lane. Load 1/2 the volume of your samples: for large combs, load 5 µl, for small combs, load 2.5 µl.

Place the lid on the gel box. Make sure it is seated on the brass contacts.

Run the gel

Press the Start or Run button on the power supply.

Make sure the power supply doesn't complain about an open circuit. If it does, re-seat the gel box lid and press Run again.

Double-check that there are bubbles forming on the platinum wires at either end of the gel box.

Double-check that the red (positive) lead is on the side farthest from the wells. Remember, Run to Red.

Run until the pink band is 2/3 to 3/4 of the way down the gel. For small analytical gels, this should take 30 minutes. For larger gels, start at 45 minutes and check regularly!

00:30:00

Image the gel

If the power supply is still running, press the Stop button.

Lift the lid off of the gel box. Lift the lid straight up. If you try to "hinge" it up, the lid will break.

Transfer the gel to the GelDoc.

If it's not running, start QuantityOne from the toolbar.

If necessary, click the top button on the toolbox to select the scanner.

If necessary, reset the camera (as per instructions on the GelDoc computer.)

Press the Epifluorescent Illumination button on the GelDoc. Check the gel's position, zoom and focus.

You want to be zoomed in so that the gel fills the field of view, and focussed so that the well edges are sharp.

Close the GelDoc door. Click Auto Expose.

The Auto Expose functionality generally over-exposes my gels a bit. If your gel is over-exposed, remove 1/3 of the exposure time, type it into the Exposure box, then click Manually Expose.

In the File menu, select, Export as JPEG.... Save your gel to the iGEM folder on the desktop.

Open Benchling. Log in, and copy the gel to the Description page for the plasmid you're building.

If there are multiple plasmids on the gel, save it to each plasmid's Description.

Discard the gel in the biowaste box. Wipe down the gel doc with a little water and a paper towel or Kimwipe.

Annotate the gel

Immediately, before you forget what's where, annotate the gel.

List what is in each lane.

Describe whether the pattern is what you expected or not. (You should have an in silico digestion to compare it to!)

Experiment 1: Restriction Digest and Gel Confirmation BM3R1 and Tal21 · Benchling

Experiment 1: Restriction Digest and Gel Confirmation BM3R1 and Tal21

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-06-22 to 2016-06-23

Wednesday, 6/22

	A	B	C	D	E	F
1	construct	enzymes	buffer	DNA concentration ng/ul (mini 1)	DNA concentration ng/ul (mini 2)	name on the PCR strip tube
2	TRE:Tal21	Stu1	CS	412.7	405.2	T:Tal21
3	TRE:BM3R1	Nhe1 + Stu1	CS	212.4	646.6	T:B
4	pBM3R1r:eYFP	Blp1 + Stu1	CS	120.7	112.5	B:E
5	pTALER21:eYFP	Blp1 + Stu1	CS	127.6	550.3	T21:E

Table1

	A	B	C	D	E	F	G	H	I
1	Cap #	Bottle Label	*all controls used mini 1
2	1	TRE:Tal21 uncut
3	2	TRE:Tal21 Stu1 mini 1
4	3	TRE:Tal21 Stu 1 mini 2
5	4	TRE:BM3R1 uncut
6	5	TRE:BM3R1 Nhe1
7	6	TRE:BM3R1 Stu1
8	7	TRE:BM3R1 DD mini 1
9	8	TRE:BM3R1 DD mini 2
10	9	BM3R1: eYFP uncut
11	10	BM3R1: eYFP Blp1
12	11	BM3R1: eYFP Stu1
13	12	BM3R1: eYFP DD mini 1
14	13	BM3R1: eYFP DD mini 2
15	14	Tal21:eYFP uncut
16	15	Tal21:eYFP Blp1
17	16	Tal21:eYFP Stu1
18	17	Tal21:eYFP DD mini 1
19	18	Tal21:eYFP DD mini 2
20

Table2

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

1

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 13

Well 14

Well 15

Well 16

Well 17

Well 18

Well 19

Well 20

2

Ladder

TRE:Tal21 uncut

TRE:Tal21 mini 1

TRE:Tal21 mini 2

TRE:BM3R1 uncut

TRE:BM3R1 Nhe1

TRE:BM3R1 Stu1

TRE:BM3R1 mini 1

TRE:BM3R1 mini 2

DNA Ladder

BM3R1:EYFP uncut

BM3R1:EYFP Blp1

BM3R1:EYFP Stu1

BM3R1:EYFP mini 1

BM3R1:EYFP mini 2

Tal21: EYFP uncut

Tal21: EYFP Blp1

Tal21: EYFP Stu1

Tal21: EYFP mini 1

Tal21: EYFP mini 2

3

Table3

xp1_062216_repressorGroup.jpg

N3200_thumb.gif

@ https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb

Thursday, 6/23

	A	B	C	D	E	F
1	construct	enzymes	buffer	DNA concentration ng/ml (mini 1)	DNA concentration ng/ml (mini 2)	name on the PCR strip tube
2	pBM3R1r:eYFP	Nco1 + Nhe1	CS	120.7	112.5	T:Tal21
3	pTALER21:eYFP	Nco1 + Stu1	CS	127.6	550.3	T:B
4	pDEST	Blp1	CS	282 (no mini!)

Table4

	A	B
1	pBM3R1r:eYFP (uncut)	B:E uncut
2	pBM3R1r:eYFP (Nco1)	B:E Nco1
3	pBM3R1r:eYFP (Nhe1)	B:E Nhe1
4	pBM3R1r:eYFP (Nco1+Nhe1)	B:E DD 1
5	pBM3R1r:eYFP (Nco1+Nhe1) mini2	B:E DD 2
6	pTALER21:eYFP (uncut)	T:E uncut
7	pTALER21:eYFP (Nco1)	T:E Nco1
8	pTALER21:eYFP (Stu1)	T:E Stu1
9	pTALER21:eYFP (Nco1+Stu1)	T:E DD 1
10	pTALER21:eYFP (Nco1+Stu1) mini2	T:E DD 2
11	pDest (uncut)	pDest uncut
12	pDest (Blp1)	pDEST cut

Table5

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

1

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 13

Well 14

Well 15

2

Ladder

BM3R1:meYFP uncut

BM3R1:meYFP Nco1

BM3R1:meYFP Nhe1

BM3R1:meYFP mini 1

BM3R1:meYFP mini 2

Ladder

Taler21:meYFP uncut

Taler21:meYFP Nco1

Taler21:meYFP Stu1

Taler21:meYFP mini 1

Taler21:meYFP mini 2

Ladder

pDest uncut

pDest Cut

3

Table6

2ndgel.PNG

Diagnostic Restriction Digestion

Introduction

A diagnostic restriction digest helps identify correctly assembled clones from incorrect clones.

Materials

200 ul PCR strip tubes, one per reaction
Restriction enzyme (chosen below), 1 ul per reaction.
10X restriction enzyme buffer (chosen below), 1 ul per reaction.

Procedure

Choose A Good Restriction Enzyme

Using Benchling, choose a restriction enzyme that meets the following criteria:

- Cuts at least once in the insert (for a pEXPR, either the promoter or the gene.)

- Cuts at least once in the backbone.

- Gives bands that aren't too large (> 8 kb) or too small (< 200 bp).

- Gives a band pattern that is significantly different from the expected error mode.

* For LR reactions, the most common error is a pDEST that slipped through the selection.

- Begin in the "Brian's Favorites" list, then expand to the main Weiss lab list.

- If you can't find a single enzyme that gives an acceptable band pattern, choose two enzymes that give a acceptable band pattern when used together.

* This double-digest is subject to buffer compatibility, outlined below.

Record your enzyme choice on the plasmid's Description page.

Benchling will tell you the enzyme's buffer compatibility and active temperature. Record the buffer in which the enzyme is most active.

If there are multiple bufers in which the enzyme is equally active, choose in this order: Buffer 3.1, Buffer CS, Buffer 2.1, Buffer 1.1.

Set up the restriction digest

Retrieve the minipreps and the appropriate 10X buffer concentrate from the freezer. Thaw on the benchtop or in your fingers.

Label the PCR tubes with your initials and an incrementing number.

ie: BT-1, -2, -3, -4

Vortex the minipreps and the 10X buffer concentrate briefly, then pulse down in the microfuge.

For each miniprep, set up a PCR tube containing the following in order:

- 5 ul enzyme-quality H2O

- 1 ul enzyme buffer

- 3 ul miniprep DNA

- 1 ul enzyme

Remove the enzyme from the freezer for as little time as possible.

I have specified an "arbitrary" 3 µl volume of miniprep DNA; this should be fine as long as your miniprep concentration is >= 100 ng/ul.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Incubate at the appropriate temperature for at least 1 hour and more more than 16 hours.

If the enzyme's active temperature is 37°C, use the 37°C plate incubator.

Stop the reaction by adding 2 ul of 6X NEB purple gel loading dye to each reacti on.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

PAUSE POINT: The reaction can be stored almost indefinitely at room temperature once it's been stopped.

Proceed to gel electrophoresis.

Gel electrophoresis

Introduction

Gel electrophoresis separates pieces of DNA by length.

Materials

Agarose gel, 1 lane per sample, plus at least 1 lane for the ladder.
- If you just poured the gel, make sure it has had at least 30 minutes to set.
- If you have a large gel but only need a few lanes, cut out just a piece of it with a razor blade. Cut straight!
Samples to run
6X NEB Purple Loading Dye
Molecular weight standard (aka "ladder")
- Commonly available ladders are Hyperladder I and NEB 2-Log.
Gel box, lid, leads
Electrophoresis power supply
1X TAE buffer, enough to fill the gel box.

Procedure

Prepare your samples

If your samples are frozen, thaw them completely, flick or vortex to mix, then pulse down in the microfuge.

If your samples are not already in loading dye, mix them 1:6 with 6X NEB Purple Loading Dye

Add 1 µl of loading dye for every 5 µl of sample.

Pro tip: If you don't want to mix loading gel with your entire sample (PCR products, for example), cut off a strip of Parafilm; pipette 2-3 ul of sample onto the parafilm; add 1 ul loading dye; mix by pipetting and load directly.

Set up the gel box

Program the voltage on the power supply. For the small gel box, use 100V; for the large gel box, use 150V.

Program the time on the power supply. For a small analytical gel, set the timer for 30 minutes.

For larger gels, start at 45 minutes, and then check regularly!

Attach the leads to the gel box cover. Make sure that the red (positive) lead is attached to the side of the box farthest away from the wells in the gel.

Remember, the DNA is negatively charged and will move toward the positive terminal. A useful neumonic for remembering this is "Run to Red."

If the gel box has not been used previously that day, empty it and rinse it out with DI water.

The TAE buffer can be re-used, but if it's been sitting out for too long it evaporates and the salt concentration (and conductivity) changes. TAE is cheap; when in doubt, replace it.

Place the gel in the gel box.

Pour TAE into the gel box until it just barely covers the gel.

The TAE's purpose is to conduct electricity; over-filling the gel box results in a larger conductive path, more current flow, and more heating (which can screw up your run.)

Check the wells to see that they are free of bubbles. If there are bubbles, blow them out by pipetting 100 µl of TAE from the gel box into the well.

Load your samples

Load your samples. For large combs, load 10 µl; for small combs, load 5 µl.

Work quickly. The samples begin to diffuse in the buffer, leading to smeared bands.

Load the ladder in the last lane. Load 1/2 the volume of your samples: for large combs, load 5 µl, for small combs, load 2.5 µl.

Place the lid on the gel box. Make sure it is seated on the brass contacts.

Run the gel

Press the Start or Run button on the power supply.

Make sure the power supply doesn't complain about an open circuit. If it does, re-seat the gel box lid and press Run again.

Double-check that there are bubbles forming on the platinum wires at either end of the gel box.

Double-check that the red (positive) lead is on the side farthest from the wells. Remember, Run to Red.

Run until the pink band is 2/3 to 3/4 of the way down the gel. For small analytical gels, this should take 30 minutes. For larger gels, start at 45 minutes and check regularly!

Image the gel

If the power supply is still running, press the Stop button.

Lift the lid off of the gel box. Lift the lid straight up. If you try to "hinge" it up, the lid will break.

Transfer the gel to the GelDoc.

If it's not running, start QuantityOne from the toolbar.

If necessary, click the top button on the toolbox to select the scanner.

If necessary, reset the camera (as per instructions on the GelDoc computer.)

Press the Epifluorescent Il lumination button on the GelDoc. Check the gel's position, zoom and focus.

You want to be zoomed in so that the gel fills the field of view, and focussed so that the well edges are sharp.

Close the GelDoc door. Click Auto Expose.

The Auto Expose functionality generally over-exposes my gels a bit. If your gel is over-exposed, remove 1/3 of the exposure time, type it into the Exposure box, then click Manually Expose.

In the File menu, select, Export as JPEG.... Save your gel to the iGEM folder on the desktop.

Open Benchling. Log in, and copy the gel to the Description page for the plasmid you're building.

If there are multiple plasmids on the gel, save it to each plasmid's Description.

Discard the gel in the biowaste box. Wipe down the gel doc with a little water and a paper towel or Kimwipe.

Annotate the gel

Immediately, before you forget what's where, annotate the gel.

List what is in each lane.

Describe whether the pattern is what you expected or not. (You should have an in silico digestion to compare it to!)

RD and GC Tal21:eyfp/Tal14:eyfp · Benchling

RD and GC Tal21:eyfp/Tal14:eyfp

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Maya Kaul

Date: 2016-07-25

Monday, 7/25

Lanes:

nothing nothing uncut pstI EcoRI Ladder pDEST A B Nothing...

Screen Shot 2016-07-25 at 7.21.01 PM.png

Screen Shot 2016-07-25 at 7.21.32 PM.png

Gel electrophoresis

Introduction

Gel electrophoresis separates pieces of DNA by length.

Materials

Agarose gel, 1 lane per sample, plus at least 1 lane for the ladder.
- If you just poured the gel, make sure it has had at least 30 minutes to set.
- If you have a large gel but only need a few lanes, cut out just a piece of it with a razor blade. Cut straight!
Samples to run
6X NEB Purple Loading Dye
Molecular weight standard (aka "ladder")
- Commonly available ladders are Hyperladder I and NEB 2-Log.
Gel box, lid, leads
Electrophoresis power supply
1X TAE buffer, enough to fill the gel box.

Procedure

Prepare your samples

If your samples are frozen, thaw them completely, flick or vortex to mix, then pulse down in the microfuge.

If your samples are not already in loading dye, mix them 1:6 with 6X NEB Purple Loading Dye

Add 1 µl of loading dye for every 5 µl of sample.

Pro tip: If you don't want to mix loading gel with your entire sample (PCR products, for example), cut off a strip of Parafilm; pipette 2-3 ul of sample onto the parafilm; add 1 ul loading dye; mix by pipetting and load directly.

Set up the gel box

Program the voltage on the power supply. For the small gel box, use 100V; for the large gel box, use 150V.

Program the time on the power supply. For a small analytical gel, set the timer for 30 minutes.

For larger gels, start at 45 minutes, and then check regularly!

Attach the leads to the gel box cover. Make sure that the red (positive) lead is attached to the side of the box farthest away from the wells in the gel.

Remember, the DNA is negatively charged and will move toward the positive terminal. A useful neumonic for remembering this is "Run to Red."

If the gel box has not been used previously that day, empty it and rinse it out with DI water.

The TAE buffer can be re-used, but if it's been sitting out for too long it evaporates and the salt concentration (and conductivity) changes. TAE is cheap; when in doubt, replace it.

Place the gel in the gel box.

Pour TAE into the gel box until it just barely covers the gel.

The TAE's purpose is to conduct electricity; over-filling the gel box results in a larger conductive path, more current flow, and more heating (which can screw up your run.)

Check the wells to see that they are free of bubbles. If there are bubbles, blow them out by pipetting 100 µl of TAE from the gel box into the well.

Load your samples

Load your samples. For large combs, load 10 µl; for small combs, load 5 µl.

Work quickly. The samples begin to diffuse in the buffer, leading to smeared bands.

Load the ladder in the last lane. Load 1/2 the volume of your samples: for large combs, load 5 µl, for small combs, load 2.5 µl.

Place the lid on the gel box. Make sure it is seated on the brass contacts.

Run the gel

Press the Start or Run button on the power supply.

Make sure the power supply doesn't complain about an open circuit. If it does, re-seat the gel box lid and press Run again.

Double-check that there are bubbles forming on the platinum wires at either end of the gel box.

Double-check that the red (positive) lead is on the side farthest from the wells. Remember, Run to Red.

Run until the pink band is 2/3 to 3/4 of the way down the gel. For small analytical gels, this should take 30 minutes. For larger gels, start at 45 minutes and check regularly!

Image the gel

If the power supply is still running, press the Stop button.

Lift the lid off of the gel box. Lift the lid straight up. If you try to "hinge" it up, the lid will break.

Transfer the gel to the GelDoc.

If it's not running, start QuantityOne from the toolbar.

If necessary, click the top button on the toolbox to select the scanner.

If necessary, reset the camera (as per instructions on the GelDoc computer.)

Press the Epifluorescent Illumination button on the GelDoc. Check the gel's position, zoom and focus.

You want to be zoomed in so that the gel fills the field of view, and focussed so that the well edges are sharp.

Close the GelDoc door. Click Auto Expose.

The Auto Expose functionality generally over-exposes my gels a bit. If your gel is over-exposed, remove 1/3 of the exposure time, type it into the Exposure box, then click Manually Expose.

In the File menu, select, Export as JPEG.... Save your gel to the iGEM folder on the desktop.

Open Benchling. Log in, and copy the gel to the Description page for the plasmid you're building.

If there are multiple plasmids on the gel, save it to each plasmid's Description.

Discard the gel in the biowaste box. Wipe down the gel doc with a little water and a paper towel or Kimwipe.

Annotate the gel

Immediately, before you forget what's where, annotate the gel.

List what is in each lane.

Describe whether the pattern is what you expected or not. (You should have an in silico digestion to compare it to!)

Diagnostic Restriction Digestion

Introduction

A diagnostic restriction digest helps identify correctly assembled clones from incorrect clones.

Materials

200 ul PCR strip tubes, one per reaction
Restriction enzyme (chosen below), 1 ul per reaction.
10X restriction enzyme buffer (chosen below), 1 ul per reaction.

Procedure

Choose A Good Restriction Enzyme

Using Benchling, choose a restriction enzyme that meets the following criteria:

- Cuts at least once in the insert (for a pEXPR, either the promoter or the gene.)

- Cuts at least once in the backbone.

- Gives bands that aren't too large (> 8 kb) or too small (< 200 bp).

- Gives a band pattern that is significantly different from the expected error mode.

* For LR reactions, the most common error is a pDEST that slipped through the selection.

- Begin in the "Brian's Favorites" list, then expand to the main Weiss lab list.

- If you can't find a single enzyme that gives an acceptable band pattern, choose two enzymes that give a acceptable band pattern when used together.

* This double-digest is subject to buffer compatibility, outlined below.

Record your enzyme choice on the plasmid's Description page.

Benchling will tell you the enzyme's buffer compatibility and active temperature. Record the buffer in which the enzyme is most active.

If there are multiple bufers in which the enzyme is equally active, choose in this order: Buffer 3.1, Buffer CS, Buffer 2.1, Buffer 1.1.

Set up the restriction digest

Retrieve the minipreps and the appropriate 10X buffer concentrate from the freezer. Thaw on the benchtop or in your fingers.

Label the PCR tubes with your initials and an incrementing number.

ie: BT-1, -2, -3, -4

Vortex the minipreps and the 10X buffer concentrate briefly, then pulse down in the microfuge.

For each miniprep, set up a PCR tube containing the following in order:

- 5 ul enzyme-quality H2O

- 1 ul enzyme buffer

- 3 ul miniprep DNA

- 1 ul enzyme

Remove the enzyme from the freezer for as little time as possible.

I have specified an "arbitrary" 3 µl volume of miniprep DNA; this should be fine as long as your miniprep concentration is >= 100 ng/ul.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Incubate at the appropriate temperature for at least 1 hour and more more than 16 hours.

If the enzyme's active temperature is 37°C, use the 37°C plate incubator.

Stop the reaction by adding 2 ul of 6X NEB purple gel loading dye to each reacti on.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

PAUSE POINT: The reaction can be stored almost indefinitely at room temperature once it's been stopped.

Proceed to gel electrophoresis.

Restriction Digestion and Gel Confirmation of TRE:BM3R1, TRE:Tal21, hef1a:Gal4VP16, Tal21:meYFP, BM3R1:meYFP · Benchling

Restriction Digestion and Gel Confirmation of TRE:BM3R1, TRE:Tal21, hef1a:Gal4VP16, Tal21:meYFP, BM3R1:meYFP

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Maya Kaul

Dates: 2016-07-05 to 2016-07-07

Tuesday, 7/5

n mTRE:BM3R1, TRE:Tal21, hef1a:Gal4VP16, Tal21:meYFP, BM3R1:meYFP

	A	B	C	D	E	F	G
1	plasmid	TRE:BM3R1	TRE:Tal21	hef1a:Gal4VP16	Tal21:meYFP	BM3R1:meYFP	pDEST_mCherry
2	enzymes	Nhe1, Stu1	Stu1	BgIII, Nco1	Nco1, Stu1	Nco1, Nhe1	Nco1, Xho1
3	label	T:B	T:T21	H:G	T:E	B:E	D:M
4
5

Table1

-100ng of DNA in each 10ul digest reaction

Calculations:

- 5 ul enzyme-quality H2O

- 1 ul enzyme buffer

- 3 ul miniprep DNA

- 1 ul enzyme

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

AB

1

LABEL

1

2

3

4

5

6

7

LABEL

8

9

10

11

12

LABEL

13

14

15

16

17

18

LABEL

19

20

21

22

23

24

2

TEMPLATE

TRE:BM3R1 - 1

TRE:BM3R1 -1

TRE:BM3R1 -3

TRE:BM3R1 -2

TRE:BM3R1 -4

TEMPLATE

TRE:Tal21 -1

TRE:Tal21 -2

TRE:Tal21 -3

TRE:Tal21 -4

TEMPLATE

hef1a: Gal4 -1

hef1a: Gal4 -2

hef1a: Gal4 -3

TEMPLATE

Tal21:EYFP -1

Tal21:EYFP -2

Tal21:EYFP -3

3

H2O

6 ul

5 ul

4 ul

H2O

6 ul

5 ul

H2O

3 ul

5 ul

4 ul

H2O

6 ul

5 ul

4 ul

4

BUFFER

1 ul

BUFFER

1 ul

BUFFER

1 ul

BUFFER

1 ul

5

DNA (100 ng/uL)

3 ul

DNA (100 ng/uL)

3 ul

DNA (100 ng/uL)

6 ul

DNA (100 ng/uL)

3 ul

6

ENZYME 1

0

1 ul nheI

0

1 ul nheI

ENZYME 1

0

1 ul stuI

ENZYME 1

0

1 ul bglII

0

1 ul bglII

ENZYME 1

0

1 ul NcoI

0

1 ul ncoI

7

ENZYME 2

0

1ul stuI

1 ul stuI

ENZYME 2

0

ENZYME 2

0

1ul ncoI

1 ul ncoI

ENZYME 2

0

1 ul stuI

Table2

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

1

LABEL

25

26

27

28

29

30

LABEL

31

32

33

34

35

36

2

TEMPLATE

BM3R1:EYFP -1

BM3R1:EYFP -2

BM3R1:EYFP -3

TEMPLATE

pDEST_mCherry -1

pDEST_mCherry -2

pDEST_mCherry -3

3

H2O

3 ul

2 ul

1 ul

H2O

6 ul

5 ul

4 ul

1 ul

4 ul

4

BUFFER

1 ul

BUFFER

1 ul

5

DNA (100 ng/uL)

6 ul

DNA (100 ng/uL)

3 uL

6 uL

3 uL

6

ENZYME 1

0

1 ul ncoI

0

1 ul ncoI

ENZYME 1

0

1 ul ncoI

0

1 ul ncoI

7

ENZYME 2

0

1 ul nheI

ENZYME 2

0

1ul xhoI

1 ul xhoI

Table3

GEL SET UP

	A	B	C	D	E	F	G	H	I	J	K	L	M	N	O	P	Q	R	S	T	U
1	LANE	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
2	TUBE	LADDER	1	2	3	4	5	6	7	8	9	10	11	12	LADDER	x	13	14	15	16	17
3	EXPECTED?	-	y	y	y	y	n	y	n	y	y	y	y	n	-	-	y	?	?	n	n
4	LANE	21	22	23	24	25	26	27	28	29	30	31	32	33	34	35	36	37	38	39	40
5	TUBE	18	19	20	21	22	23	24	LADDER	25	26	27	28	29	30	31	32	33	34	35	36
6	EXPECTED?	n	?	n	n	n	n	n	-	?	n	n	n	n	n	n	n	n	n	n	n

Table4

Thursday, 7/7

Sequencing

	A	B	C
1	Tube	DNA Template	Results
2	1	TRE:BM3R1 -2	fine
3	2	TRE:BM3R1 -2	-
4	3	TRE:Tal21 -1	shaky sequence but probably ok
5	4	TRE:Tal21 -1	-
6	5	hef1a:gal4 -1	fine
7	6	hef1a:gal4 -1	-
8	7	Tal21:eyfp -1	fine, **eyfp instead of mEYFP
9	8	Tal21:eyfp -1	-
10	9	BM3R1: EYFP -3	mostly fine, eyfp not efp. can ask about weirdness at att B2
11	10	BM3R1: EYFP -3	-
12	11	pDEST_mCherry -1	mCherry not there --> problem with purification or digestion sites?
13	12	pDEST_mCherry -1	-

Table5

pDEST-mCherry -- Notes from Brian

AsiSI is an enzyme Brian hasn't had experience with --> May be cause?

Could the purification

Do PCR again, scale to 50 uL. 2.5 uL primer.... Try gel purification

Cut for longer.. 2 hours

Likely don't need to purify

Diagnostic Restriction Digestion

Introduction

A diagnostic restriction digest helps identify correctly assembled clones from incorrect clones.

Materials

200 ul PCR strip tubes, one per reaction
Restriction enzyme (chosen below), 1 ul per reaction.
10X restriction enzyme buffer (chosen below), 1 ul per reaction.

Procedure

Choose A Good Restriction Enzyme

Using Benchling, choose a restriction enzyme that meets the following criteria:

- Cuts at least once in the insert (for a pEXPR, either the promoter or the gene.)

- Cuts at least once in the backbone.

- Gives bands that aren't too large (> 8 kb) or too small (< 200 bp).

- Gives a band pattern that is significantly different from the expected error mode.

* For LR reactions, the most common error is a pDEST that slipped through the selection.

- Begin in the "Brian's Favorites" list, then expand to the main Weiss lab list.

- If you can't find a single enzyme that gives an acceptable band pattern, choose two enzymes that give a acceptable band pattern when used together.

* This double-digest is subject to buffer compatibility, outlined below.

Record your enzyme choice on the plasmid's Description page.

Benchling will tell you the enzyme's buffer compatibility and active temperature. Record the buffer in which the enzyme is most active.

If there are multiple bufers in which the enzyme is equally active, choose in this order: Buffer 3.1, Buffer CS, Buffer 2.1, Buffer 1.1.

Set up the restriction digest

Retrieve the minipreps and the appropriate 10X buffer concentrate from the freezer. Thaw on the benchtop or in your fingers.

Label the PCR tubes with your initials and an incrementing number.

ie: BT-1, -2, -3, -4

Vortex the minipreps and the 10X buffer concentrate briefly, then pulse down in the microfuge.

For each miniprep, set up a PCR tube containing the following in order:

- 5 ul enzyme-quality H2O

- 1 ul enzyme buffer

- 3 ul miniprep DNA

- 1 ul enzyme

Remove the enzyme from the freezer for as little time as possible.

I have specified an "arbitrary" 3 µl volume of miniprep DNA; this should be fine as long as your miniprep concentration is >= 100 ng/ul.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Incubate at the appropriate temperature for at least 1 hour and more more than 16 hours.

If the enzyme's active temperature is 37°C, use the 37°C plate incubator.

Stop the reaction by adding 2 ul of 6X NEB purple gel loading dye to each reacti on.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

PAUSE POINT: The reaction can be stored almost indefinitely at room temperature once it's been stopped.

Proceed to gel electrophoresis.

Gel electrophoresis

Introduction

Gel electrophoresis separates pieces of DNA by length.

Materials

Agarose gel, 1 lane per sample, plus at least 1 lane for the ladder.
- If you just poured the gel, make sure it has had at least 30 minutes to set.
- If you have a large gel but only need a few lanes, cut out just a piece of it with a razor blade. Cut straight!
Samples to run
6X NEB Purple Loading Dye
Molecular weight standard (aka "ladder")
- Commonly available ladders are Hyperladder I and NEB 2-Log.
Gel box, lid, leads
Electrophoresis power supply
1X TAE buffer, enough to fill the gel box.

Procedure

Prepare your samples

If your samples are frozen, thaw them completely, flick or vortex to mix, then pulse down in the microfuge.

If your samples are not already in loading dye, mix them 1:6 with 6X NEB Purple Loading Dye

Add 1 µl of loading dye for every 5 µl of sample.

Pro tip: If you don't want to mix loading gel with your entire sample (PCR products, for example), cut off a strip of Parafilm; pipette 2-3 ul of sample onto the parafilm; add 1 ul loading dye; mix by pipetting and load directly.

Set up the gel box

Program the voltage on the power supply. For the small gel box, use 100V; for the large gel box, use 150V.

Program the time on the power supply. For a small analytical gel, set the timer for 30 minutes.

For larger gels, start at 45 minutes, and then check regularly!

Attach the leads to the gel box cover. Make sure that the red (positive) lead is attached to the side of the box farthest away from the wells in the gel.

Remember, the DNA is negatively charged and will move toward the positive terminal. A useful neumonic for remembering this is "Run to Red."

If the gel box has not been used previously that day, empty it and rinse it out with DI water.

The TAE buffer can be re-used, but if it's been sitting out for too long it evaporates and the salt concentration (and conductivity) changes. TAE is cheap; when in doubt, replace it.

Place the gel in the gel box.

Pour TAE into the gel box until it just barely covers the gel.

The TAE's purpose is to conduct electricity; over-filling the gel box results in a larger conductive path, more current flow, and more heating (which can screw up your run.)

Check the wells to see that they are free of bubbles. If there are bubbles, blow them out by pipetting 100 µl of TAE from the gel box into the well.

Load your samples

Load your samples. For large combs, load 10 µl; for small combs, load 5 µl.

Work quickly. The samples begin to diffuse in the buffer, leading to smeared bands.

Load the ladder in the last lane. Load 1/2 the volume of your samples: for large combs, load 5 µl, for small combs, load 2.5 µl.

Place the lid on the gel box. Make sure it is seated on the brass contacts.

Run the gel

Press the Start or Run button on the power supply.

Make sure the power supply doesn't complain about an open circuit. If it does, re-seat the gel box lid and press Run again.

Double-check that there are bubbles forming on the platinum wires at either end of the gel box.

Double-check that the red (positive) lead is on the side farthest from the wells. Remember, Run to Red.

Run until the pink band is 2/3 to 3/4 of the way down the gel. For small analytical gels, this should take 30 minutes. For larger gels, start at 45 minutes and check regularly!

Image the gel

If the power supply is still running, press the Stop button.

Lift the lid off of the gel box. Lift the lid straight up. If you try to "hinge" it up, the lid will break.

Transfer the gel to the GelDoc.

If it's not running, start QuantityOne from the toolbar.

If necessary, click the top button on the toolbox to select the scanner.

If necessary, reset the camera (as per instructions on the GelDoc computer.)

Press the Epifluorescent Illumination button on the GelDoc. Check the gel's position, zoom and focus.

You want to be zoomed in so that the gel fills the field of view, and focussed so that the well edges are sharp.

Close the GelDoc door. Click Auto Expose.

The Auto Expose functionality generally over-exposes my gels a bit. If your gel is over-exposed, remove 1/3 of the exposure time, type it into the Exposure box, then click Manually Expose.

In the File menu, select, Export as JPEG.... Save your gel to the iGEM folder on the desktop.

Open Benchling. Log in, and copy the gel to the Description page for the plasmid you're building.

If there are multiple plasmids on the gel, save it to each plasmid's Description.

Discard the gel in the biowaste box. Wipe down the gel doc with a little water and a paper towel or Kimwipe.

Annotate the gel

Immediately, before you forget what's where, annotate the gel.

List what is in each lane.

Describe whether the pattern is what you expected or not. (You should have an in silico digestion to compare it to!)

LR Reaction

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

Promoter pENTR plasmid: L4-Promoter-R1
- Working concentration: 5 fmol/ul
Gene pENTR plasmid: L1-Gene-L2
- Working concentration: 5 fmol/ul
Destination plasmid: pDEST
- Working concentration: 10 fmol/ul
Nuclease-free TE
200 µl PCR strip tubes, 1 tube per rxn
5x LR Clonase II
- Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
Proteinase K
- Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

LR Reaction Setup

For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label
2	Promoter pENTR
3	Gene pENTR
4	pDEST
5
6

Table1

For each LR, label a 200 µl strip tube with your initials and tube number.

Into each tube, pipette:

-- 1 µl of the promoter pENTR

-- 1 µl of the gene pENTR

-- 1 µl of the pDEST

Add 1 µl of TE to each tube

Retrieve an aliquot of LR Clonase from the -80.

Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

Add 1 µl of the LR clonase to each LR reaction.

Be careful pipetting; LR clonase is viscous.

Cap the tubes.

Flick them several times to mix.

Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

Incubate at room temperature for at least 12 hours and not more than 24 hours.

A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

16-24 hours later: Proteinase K kill

Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.m n

Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

Pipette 1 ul into each of the LR reactions.

Flick several times to mix.

Pulse-spin the tubes in the microcentrifuge.

Incubate at 37° for 15 minutes, or room-temperature for an hour.

PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

00:15:00

Proceed to transformation. Transform 2 µl.

Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

Team:MIT/Repressor Group Experiment 1 Digestion Gels

Experiment 1 Restriction Digestion for testing

digestcalculations.jpg

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Diagnostic Restriction Digestion (Maya, Liz, Wangui, Kathryn 4/18/16)

Introduction

Materials

Procedure

Pouring an agarose gel

Introduction

Materials

Procedure

Gel electrophoresis

Introduction

Materials

Procedure

Experiment 1: Restriction Digest and Gel Confirmation BM3R1 and Tal21

xp1_062216_repressorGroup.jpg

N3200_thumb.gif

2ndgel.PNG

Diagnostic Restriction Digestion

Introduction

Materials

Procedure

Gel electrophoresis

Introduction

Materials

Procedure

RD and GC Tal21:eyfp/Tal14:eyfp

Screen Shot 2016-07-25 at 7.21.01 PM.png

Screen Shot 2016-07-25 at 7.21.32 PM.png

Gel electrophoresis

Introduction

Materials

Procedure

Diagnostic Restriction Digestion

Introduction

Materials

Procedure

Restriction Digestion and Gel Confirmation of TRE:BM3R1, TRE:Tal21, hef1a:Gal4VP16, Tal21:meYFP, BM3R1:meYFP

Diagnostic Restriction Digestion

Introduction

Materials

Procedure

Gel electrophoresis

Introduction

Materials

Procedure

LR Reaction

Introduction

Materials

Procedure