Team:MIT/Recombinase Group Notebook Week 9

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
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• NOTEBOOK
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• AWARDS
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• TEAM

TN 08/01/16

Made with Benchling

Project: iGem 2016 _ Recombinase subgroup

Authors: Trinh Nguyen

Date: 2016-08-01

Monday, 8/1

Transfection set-up

Plate Map

	A	B	C	D
1	Untransfected control	Single color (Y) 250ng hEF1a:eYFP 250ng pDONR	Single color (R) 250ng hEF1a:mKate 250ng pDONR	Two color 250ng hEF1a:eYFP 250ng hEF1a:mKate
2	Experimental 1 250ng hEF1a:SV40:eYFP 250ng hEF1a:mKate	Experimental 2 250ng hEF1a:flipped eYFP 250ng hEF1a:mKate

Table1

Transfection mixture

Before preparing the transfection mixture, make a dilution of pDONR (200ng/uL) to 100ng/uL.

	A	B	C	D	E	F	G
1	uL	Single color (Y)	Single color (R)	Two color	Experimental 1	Experimental 2
2	DNA 1	2.5 EYFP	2.5 mKate	2.5 eYFP	2.5 SV40:eYFP	2.5 flipped eYFP
3	DNA 2	2.5 pDONR	2.5 pDONR	2.5 mKate	2.5 mKate	2.5 mKate
4	Viafect	1	1	1	1	1
5	Opt Mem	44	44	44	44	44
6	Total	50	50	50	50	50

Table2

DNA Transfection into HEK Cells

Introduction

Get started by giving your protocol a name and editing this introduction.

Materials

• OptiMEM (Serum-free media)
• Viafect
• DNA from midipreps

Procedure

• Phase I: Calculations

1. Select DNA to be transfected and determine the concentration through Nanodrop

2. Determine the volume of DNA required to have 0.5 ug in reaction mixture.

• Ex. for a 521 ng/ul stock of DNA you would need approximately 1 uL. If the calculated volume you need is less than 0.5 uLr (i.e. hard to pipette) you may want to dilute your DNA solution so that you can get an appropriate volume.

3. Determine the amount of Viafect to use from the following table:

	A	B	C	D	E	F	G	H
1	Volume of cells in media (per well)	Total volume transfection complex (per well)	Amount of DNA (per well)	Amt of Viafect Reagent for 1.5:1 Reagent:DNA	2:1	3:1	4:1	6:1
2	500 uL	50 uL	0.5 ug	0.75 uL	1 uL	1.5 uL	2 uL	3 uL

Table1

4. Calculate the amount of serum free DMEM media required to get a reaction volume of 50 uL

• Ex. if you are adding 1 uL of DNA and 3 uL of Viafect, you would want to add 46 uL of DMEM

• Phase II: Reaction Mixture

5. This phase consists of mixing the reactants required to create the transfection complex. Do so for all ratios of reagent to DNA above - add media first, then reagent, then DNA.

6. Let the reaction mixture sit for 10-20 minutes

• Closer to 20 min is better

• Phase III: Transfection

7. Retrieve cells from incubator. Make sure to leave this step for last because you don’t want to keep the cells at room temperature for too long.

8. Add entire 50 uL transfection complex to each well in the well plate and store your cells back in the incubator.

• Phase IV: Checking for Fluorescence

9. Close off lights in room and turn on the microscope’s light.

10. Apply the appropriate fluorescence filter.

11. Check for fluorescence.

CF 08/02/16

Made with Benchling

Project: iGem 2016 _ Recombinase subgroup

Authors: Elizabeth Strand

Date: 2016-08-02

Tuesday, 8/2

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

1. 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	pERE3	pERE5	pERE6
3	Gene pENTR	TP901	TP901	TP901
4	pDEST	pDEST_mCherry	pDEST_mCherry	pDEST_mCherry
5
6

Table1

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

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 1 µl of the pDEST

4. Add 1 µl of TE to each tube

5. 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.

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

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

• Be careful pipetting; LR clonase is viscous.

8. Cap the tubes.

9. Flick them several times to mix.

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

11. 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

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

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

14. Pipette 1 ul into each of the LR reactions.

15. Flick several times to mix.

16. Pulse-spin the tubes in the microcentrifuge.

17. 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

18. Proceed to transformation. Transform 2 µl.

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

JG 8/03/16

Made with Benchling

Project: iGem 2016 _ Recombinase subgroup

Authors: Elizabeth Strand

Date: 2016-08-03

Wednesday, 8/3

Transfection experiment set up

Plate map

	A	B	C	D
1	Untransfected control	Single color (Y) 500ng hEF1a:eYFP 500ng pDONR	Single color (R) 500ng hEF1a:mKate 500ng pDONR	Single color (B) 500ng hEF1a:BFP 500ng pDONR
2	Three colors 300ng hEF1a:eYFP 300ng hEF1a:mKate 300ng hEF1a:BFP 100ng pDONR	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 0nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 20 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 50 nM Dox
3	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 100 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 200 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 500 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 1000 nM Dox
4	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:BFP 200ng pDONR 2000 nM Dox	200ng hEF1a:flp-eYFP 300ng EGSH:TP901 100ng hEF1a:VgEcr 100ng hEF1a:BFP 300ng EGSH:mKate 0uM PonA	200ng hEF1a:flp-eYFP 300ng EGSH:TP901 100ng hEF1a:VgEcr 100ng hEF1a:BFP 300ng EGSH:mKate 0.5uM PonA	200ng hEF1a:flp-eYFP 300ng EGSH:TP901 100ng hEF1a:VgEcr 100ng hEF1a:BFP 300ng EGSH:mKate 1uM PonA
5	200ng hEF1a:flp-eYFP 300ng EGSH:TP901 100ng hEF1a:VgEcr 100ng hEF1a:BFP 300ng EGSH:mKate 5uM PonA

Table1

Mixture preparation: L7Ae toxicity

	A	B	C	D
1		Amount (uL)	Amount (ng)	8 samples
2	TRE:L7Ae	3	300	24
3	TRE:mKate	3	300	24
4	hEF1a:rtTA	1	100	8
5	hEF1a:BFP	1	100	8
6	pDONR	2	200	16
7	Viafect	2		16
8	Opti MEM	38		304
9	Total	50	1000

Table2

TP901 flipped eYFP

	A	B	C	D
1		Amount (uL)	Amount (ng)	4 samples
2	hEF1a:flp-eYFP	2	200	8
3	EGSH:TP901	3	300	12
4	EGSH:mKate	3	300	12
5	hEF1a:VgEcr	1	100	4
6	hEF1a:BFP	1	100	4
7	Viafect	2		8
8	Opti MEM	38		152
9	Total	50	1000

Table3

Controls

	A	B	C	D	E
1		Single (Y)	Single (R)	Single (B)	Three
2	hEF1a:eYFP	5	0	0	3
3	hEF1a:mKate	0	5	0	3
4	hEF1a:BFP	0	0	5	3
5	pDONR	5	5	5	1
6	Viafect	2	2	2	2
7	Opti MEM	38	38	38	38
8	Total	50	50	50	50

Table4

Transformation - Plating optimization for pDEST-mCherry

1 (standard for all 3 LRs) - streak transformation product

2 - streak 50% dilution in water

3 - Golden Gate plating protocol - 200uL water + 10 uL transformation + beads

4 - 200 uL water + 5 uL transformation + beads

Pouring LB Agar Plates

Introduction

The media must be autoclaved before you pour plates. This is not something for iGEMers to do; ask an advisor.

Materials

• One sleeve of empty plates per 500 ml of media
  • Make sure you are using the Weiss lab's bacteriological plates, not tissue culture plates.
• LB-Agar media, either solid or already melted
• 1000X stock solution of the antibiotic
  • (Amp)icillin, (Kan)amycin, (Chlor)amphenicol

Procedure

• Making media

1. Make sure you don't already have a bunch of the plates already in the fridge

2. Fill a 1-liter bottle with 500 ml of water.(You can make up to 900mL for a 1-L bottle)

3. Weigh out XXXX of powdered LB-Agar (check the bottle for the weight.)

• IF YOU MAKE A MESS OF THE WEIGH STATION, CLEAN UP AFTER YOURSELF.

4. Ask an instructor to autoclave your media.

• Melting media

5. Solid media must be melted in the microwave. Be careful -- it gets very hot.

6. Loosen the cap to prevent the bottle from exploding. 1 full turn of the cap is sufficient.

7. Microwave 2-3 minutes, watching carefully, until the media begins to melt and bubble.

8. Continue microwaving in short bursts until the media is completely melted.

• IF YOU MAKE A MESS OF THE MICROWAVE, CLEAN UP AFTER YOURSELF.

9. Cool the media to <= 60°C before adding the antibiotics.

• You can either swirl it under a cold tap until you can handle it without burning yourself, or you can leave it for a few hours in the 55°C water bath.

• To check the media temperature, swirl the bottle, wait ~30 seconds and measure with an IR thermometer. (Pew pew.)

• Pouring plates

10. Add antibiotic.

• Unless otherwise noted, it is 1000X -- so for 500 ml of media, add 500 µl of antibiotic.

• Note: If you're pouring Golden Gate plates, XGal is 500X! (for 500 ml of media, add 1000 µl of XGal)

11. Pour all of the media you add these too. Refreezing and thawing has unpredictable results on the amount of antibiotic destroyed by the heat, so to avoid over/under dosing don't refreeze. 500 ml makes a sleeve of plates.

12. Tighten the lid to avoid spills and swirl the bottle to mix

13. Use a seriological pipette to add 20ml to each plate.

• Use sterile practices

• If you get bubbles, use the pipette to try and suck the air out of the bubbles, this removes them

• 20ml is a rough estimate, what is important is that you get a solid layer with no holes.

14. Mark the plates with a stripe of marker up the side, based on which antibiotic(s) you added:

• Blue: Amp

• Red: Kan

• Green: Cm

• Black: Non-antibiotic additive (like X-gal)

15. Dry on the bench overnight.

16. Re-stack the plates upside-down and label the sleeve with the antibiotics, your initials and the date.

Transformation of E. coli

Introduction

Transformation is the process of inducing chemically competent E. coli to take up DNA.

Materials

• Dry bath, set to 42°C
  • Fill the wells in the dry bath block 1/2 full with DI water.
• Ice bucket, with ice
  • For thawing competent cells.
• DNA to transform
  • Could be an assembly reaction (LR, Golden Gate, etc) or a miniprepped plasmid.
  • If you removed it from the freezer, make sure it's entirely thawed out.
• pUC19 Transformation Control, 1 pg/µl
  • The pUC19 control will tell you how efficient your transformations were.
• SOC growth media, at room temperature
  • Check to make sure it's clear and NOT CLOUDY.
• Antibiotic plates, one per transformation, plus 1 Amp plate for the pUC19 control
  • Make sure the plates you use match the resistance cassette of the plasmid!
• Competent E. coli, one tube per transformation + one for the pUC19 control
  • These live in the -80 in 235.
  • Thaw on ice 3-4 minutes.
• A timer, set for 30 seconds.

Procedure

• Setup

1. Make sure the dry bath is set to 42°C and the wells in the block are 1/2 full of DI water

2. Remove selection plates from the refrigerator. Double-check that they match the selection marker on your plasmid, then place them in the 37° incubator.

3. Retrieve the DNA to transform.

• If frozen: thaw, completely, flick a few times to mix, then pulse down in the microfuge.

4. Fill an ice bucket with ice. Retrieve one tube of competent E. coli per transformation from the -80 and thaw on ice, 3-4 minutes.

00:03:00

5. While the transformation tubes are thawing, label their tops with something descriptive. Record the labels here:

	A	B	C	D	E	F	G	H
1	puc-19L
2	puc-19TC
3	Pdest_mCherry
4	pDest

Table1

• Transformation

6. Add 2 µl DNA from each reaction to a tube of competent cells.

• Immediately after adding the DNA to each tube, stir the cells a few times with the pipette tip.

7. Add 1 µl of the pUC19 transformation control to the positive control tube.

8. Incubate on ice for 30 minutes.

00:30:00

9. Heat shock the cells for exactly 30 seconds in the 42° heat block. (Yes, set a timer.)

00:00:30

10. Place back on ice for 2 minutes.

00:02:00

11. Add 250 µl SOC to each tube.

12. Tape the tubes to the platform of a shaker at 37°C and shake at 270 RPM for 60 minutes.

01:00:00

• Plating

13. Label the selection plates using the labels you recorded above.

14. Shake ~10 plating beads onto each plate.

15. Pipette 100 µl of each transformation onto the corresponding plates. NOTE: if you are using DNA from a golden gate reaction, see the golden gate protocol for instructions to dilute your sample.

16. -----Golden Gate: Plate only 10 ul of the outgrowth in a 200 ul puddle of water, or you will get a lawn of colonies.

17. Cover the plates and shake the beads around to spread the cells out.

18. Dispose of the beads by tapping them into the waste container.

19. Incubate the plates upside down overnight in the 37° incubator.

• Don't incubate for more than 18-24 hours.

• If LR was Done using pDEST_mCherry

20. When plating, use the plating wands to streak the plates. The plating wands are blue and should be found in the next to the PCR and eppendorf tubes in our bench.

• Compute transformation efficiency

21. Count the colonies on your positive transformation plate.

• If there are many many colonies, then hooray! You had a great transformation. Just estimate.

22. Divide the number of colonies by the fraction of the transformation you plated.

• So, if you resuspended your transformation in a total volume of 300 ul, then plated 100 ul, multiply the number of colonies by 3.

23. Transformation efficiency is expressed in colonies per microgram pUC19. Multiply the number of colonies by the appropriate conversion factor.

• So if you transformed 1 picogram of pUC19 DNA, multiply by 10⁶.

24. Record your transformation efficiency in your (daily) lab notebook.

CF 08/04/16

Made with Benchling

Project: iGem 2016 _ Recombinase subgroup

Authors: Elizabeth Strand

Date: 2016-08-04

Thursday, 8/4

	A	B
1	1	Q2-SloxP-Q3
2	2	Q4-SloxP-Qx
3	3	Q2-FRT-Q3
4	4	Q4-FRT-Qx
5	5	Q2-kturn-Q3
6	6	Q3-kturn-Qx
7	7	Q3-kturn-Q4
8	8	Q4-kturn-Q5
9	9	Q5-kturn-Qx

Table1

Resuspending Primers

Introduction

Primers arrive from IDT dehydrated; to use them, you need to resuspend them in TE to a known concentration. By convention, the stock concentration (in the blue-capped IDT tube) is 100 µM; the working concentration depends on the application: - For PCR, the working concentration is usually 10 µM - For sequencing, the working concentration is usually 5 µM

Materials

• Nuclease-free TE
  • We resuspend in TE to help supress nuclease activity that would degrade the primers.
• Dehydrated primers from IDT
• One extra epi tube per primer
  • ...for the working stock.

Procedure

• Resuspend the dried primer

1. Label the top of the oligo tubes. I recommend the group initials and a number.

2. In the little microfuge, spin the (dry) oligos briefly.

• Sometimes the freeze-dried primers flake off the bottom of the tube.

3. Determine how many nanomoles of primer are in the tube.

• This is the bottom line on the tube label. As an example, the BT-01 primer says

• 7.3OD = 33.4 nmol = 0.23 mg. <-- the number of nanomoles is "33.4".

4. Add 10 µl of nuclease-free TE for each nanomole of primer.

• For the BT-01 primer, this volume is 334 µl.

5. Vortex briefly.

6. Check the bottom of the tube to see if the primer is fully resuspended. If not, vortex again.

7. Pulse spin the primers.

• Make a working stock

8. Label the top of the epi tube the same as the oligo.

9. Determine the working stock concentration that you want. Label the side of the epi tube with the working concentration.

10. Make 100 µl of working stock.

• For example, if you're making a 10 µM working stock, dilute 10 µl of the concentrated stock into 90 µl of TE.

11. Vortex briefly.

12. Freeze both the concentrated and working stocks in the primers box.

Annealing Oligos

Introduction

Anealing Oligos

Materials

• Nuclease-free TE
  • We resuspend in TE to help supress nuclease activity that would degrade the primers.
• Annealing buffer: TE + 50 mM NaCl
• Dehydrated oligos from IDT
• One extra epi tube per oligo
• One thin-walled 200 ul PCR tube

Procedure

• Preparing stock solution

1. Briefly spin each tube BEFORE opening

2. Add sterile 1X TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) in a volume of uL equal to 10 times the nmol amount listed

• Concentration of stock solution is 100 uM

3. Store at -20 ^oC

• Procedure

4. Set a thermocycler to a block temperature of 95°C and a lid temperature of 105°C.

5. Dilute oligo stock solutions to 5 uM working stock (95 uL TE + 5 uL of 100 uM stock)

6. Combine 98 uL of annealing buffer, and 1 uL of 5 uM forward primer, and 1 uL of 5 uM reverse primer in a thin-walled PCR tube. Mix well.

• The concentration for the anealed oligos is 50 nM (50 fmol/uL)

7. Place tube in a standard heatblock at 90–95 °C for 3–5 minutes.

8. Remove the heat block from the apparatus and allow to cool to room temperature (or at least below 30 °C) on the workbench. Slow cooling to room temperature should take 45–60 minutes.

9. Store on ice or at 4 °C until ready to use.

Overnight liquid cultures (picking colonies)

Introduction

Overnight cultures are used to prepare miniprep DNA.

Materials

• The plate from which you are picking colonies
• 15 ml round-bottom polystyrene tubes, one per culture
  • The ones with the snap caps, NOT conical tubes with screw caps
• 5 mL LB per culture
• A container that can hold 5ml x the number of cultures
  • For a modest number of minipreps, a 50 ml conical tube works well.
  • For larger minipreps, use a sterile bottle (100 ml is frequently useful.)
• Antibiotic stock, 1000X

Procedure

• Materials Setup

1. Warm up the LB to at least room temperature (if it came from the fridge), but not warmer than 37°C

2. Label one round-bottom culture tube for each miniprep. Use "NAME-1, NAME-2, ..."etc for the naming convention, where NAME is a shortened name of the plasmid (eg, "hEF1a:mKate").

• Your impulse is to just use number, or initials and number, but trust me -- you will want to be able to identify this tube in three weeks when you've forgotten what you were doing.

3. Using a sterile pipette, transfer 5 ml of LB to the mixing container for each culture PLUS 5 ML.

4. Add antibiotic stock to a final concentration of 1X (1 µl stock for each 1 ml in the mixing container.)

5. Cap tightly and mix well.

• Culture Setup

6. Using a sterile pipette, transfer 5 ml of LB+antibiotic to each round-bottom culture tube.

• If you are making cultures with different antibiotics, take care that the right media goes in each tube.

7. Squirt ethanol on a pair of foreceps and wipe dry with a Kimwipe.

8. Use the foreceps to pick up a sterile 200µl pipette tip, scrape a colony off of the plate, and drop the pipette tip in the corresponding tube.

• Repeat for each tube.

9. Transfer to an incubating shaker at 37°C and incubate 14-16 hours.

• Don't over-grow too badly, or your yield will suffer.

• If you need to grow longer, you can grow at 30°C instead for 20 hours.

DB 08/05/16

Made with Benchling

Project: iGem 2016 _ Recombinase subgroup

Authors: Elizabeth Strand

Date: 2016-08-04

Thursday, 8/4

3-1 pERE6:TP901 - 376.8 ng/uL

1-3 pERE3:TP901 - 492.9 ng/uL

2-2 pERE5:TP901 - 512.7 ng/uL

RecombinaseGel.jpg

Digest 8:5.png

clipboard_2016-08-05_11:01:09.png

Miniprep

Introduction

The miniprep uses silica gel to isolate plasmid DNA from an E. coli culture

Materials

• Buffer P1 (resuspension buffer)
  • Retrieve from refrigerator. If you are opening a new miniprep kit, add the RNAse and LyseBlue reagent and check the box on the cap.
• Buffer P2 (lysis buffer)
  • Open the cap and look at the lysis buffer. Swirl it around. If it appears cloudy, the SDS has fallen out of solution; warm it for a few minutes in the 55°C water bath.
• Buffer N3 (neutralization buffer)
• Buffer PB (binding buffer)
• Buffer PE (rinse buffer)
  • Make sure the "Ethanol added?" box has been checked. If you are opening a new miniprep kit, add absolute ethanol as per the kit instructions and check the box on the cap.
• Buffer EB (elution buffer)
• Miniprep waste container
  • Miniprep buffers contain salts that can't go down the sink.
• Per miniprep: two microcentrifuge tubes and one blue spin column, with collection tube.

Procedure

• Harvest and resuspension

1. For each culture, label two microcentrifuge tubes on the cap and one blue spin column on the side.

• The spin columns should be in their (cap-less) collection vials.

2. Pipette 1.6 mL of each culture into the corresponding microcentrifuge tubes.

• (This is for a 1.7 ml microcentrifuge tube; set your pipettor to 800 ul and do two transfers. If we ever go back to 2-ml tubes, do 1.9 ml (2x950 ul))

3. Centrifuge at maximum speed (10,000 or 13,000xg) for three minutes.

4. Aspirate the supernatant, or pour it off into the bleach bucket.

5. Pipette ANOTHER 1.8 ml of each culture into the corresponding microcentrifuge tubes.

6. Centrifuge at maximum speed for three minutes.

7. While the centrifuge is running, move the remaining cultures to 4degC.

8. Aspirate the supernatant off with the bench aspirator. Be careful not to disturb the pelleted E. coli.

• We use an aspirator here because the less extra salt and protein we put in the miniprep, the better the yield is.

9. Add 250 µl Buffer P1 to each tube.

10. Resuspend the E. coli pellet. The preferred way is with the roto-mixer at the other end of the lab.

• Alternately, if you have just a few tubes, you can resuspend on a vortex.

• Make sure to resuspend fully and thoroughly. The resulting suspension should be smooth and cloudy; if there is particulate matter floating around, vortex some more.

• Lysis

11. Add 250 µl Buffer P2 to each tube.

• Work quickly; the lysis step should take less than 5 minutes.

12. Snap the tubes closed and invert them 4-6 times, until the tube is thoroughly mixed and the entire solution turns blue.

• If you have many many tubes, you can stack a second tube rack on top of them and invert the entire thing.

13. Add 350 ul Buffer N3 to each tube.

14. Snap the tubes closed and invert 4-6 times, until the solution is thouroughly mixed and no longer blue.

• The solution will become cloudy or flocculent.

15. Centrifuge on high speed for 10 minutes.

• Separation

16. Remove the tubes from the microcentrifuge, being careful not to disturb the white pellet.

17. Using P-1000 micropipettor set to 850 ul, carefully transfer the supernatant from each centrifuge tube to the corresponding blue spin column.

18. Centrifuge the spin columns for 30 seconds at maximum speed.

• Don't forget to put the lid on the rotor! Some of the salts get aerosolized because the spin columns don't have caps.

19. Pour the flow-through from each column into the miniprep waste container.

20. Pipette 500 ul of Buffer PB onto each spin column.

21. Centrifuge the spin columns for 30 seconds at maximum speed.

22. Pour the flow-through from each column into the miniprep waste container.

23. Pipette 750 ul of Buffer PE onto each spin column

24. Wait 1-3 minutes.

• This allows some of the salt that's still bound to the silica matrix to resuspend in the buffer.

25. Centrifuge the spin columns for 30 seconds at maximum speed.

26. Pour the flow-through from each column into the miniprep waste container.

27. Return each spin column to its collection tube and centrifuge an additional 1 minute at high speed.

• This removes every last trace of buffer PE; the ethanol can screw up downstream steps.

28. Transfer each spin column to a clean labelled microcentrifuge tube.

29. Pipette 50 ul of Buffer EB onto the center of each column.

• The volume of EB is comparable to the volume of silica gel matrix; if you pipette down the side, you might not get the entire transfer to the matrix.

30. Wait 1-3 minutes.

• This gives the DNA a chance to dissociate from the silica matrix.

31. Centrifuge the spin columns, in their collection tubes, for one minute at maximum speed.

32. Proceed directly to analyze the samples on the Nanodrop.

Golden Gate Assembly

Introduction

Golden Gate is an assembly method using Type II-S restriction enzymes and predefined overlap sequences to assemble multiple pieces of DNA in one reaction. Golden Gate design is beyond the scope of this protocol; this protocol is the reaction setup only.

Materials

• 200 µl thin-wall PCR strip tubes, one per reaction
• Backbone plasmid
  • Usually pDONR-GG L1_lacZ_L2 or pDONR-GG L4_lacZ_R1
• Parts being assembled, either in plasmids or gBlocks
• Nuclease-free water
• T4 ligase buffer, 1.5 µl per reaction
  • Found in single-use aliquots of 10 (??) µl in the -20° freezer. T4 ligase buffer does NOT like to be thawed and frozen again; once you use an aliquot, discard what's left!
• 10X BSA, 1.5 µl per reaction
  • Found in the -20° freezer. ...and that's Bovine Serum Albumin, NOT the BsaI enzyme!
• BsaI (or AarI) restriction enzyme, 1 µl per reaction
  • Leave the BsaI and T4 ligase in the freezer until the very end, and add after the rest of the reaction has been set up.
• T4 ligase, 1 µl per reaction
  • Many protocols (both online and in the lab) call for high-concentration T4 ligase. This is only necessary if you're doing VERY LARGE Golden Gate reactions (5-8 parts). You do NOT need the HC ligase for most of the GGs you'll be doing.
  • Leave the BsaI and the T4 ligase in the freezer until the very end, and add after the rest of the reaction has been set up.
• An empty 10µl or 200µl tip box
• Ice in an ice bucket

Procedure

• Record your reaction setup.

1. A Golden Gate works best when there are equimolar amounts of each part in the reaction. By convention, we use 50 fmol of each part. For each part (plasmid and gBlock), compute the concentration (fmol/ul) and the volume required for 50 fmol in the table below. If you resuspended your gBlocks at 50 fmol/ul, you can omit them in this table.

	A	B	C	D	E
1	Part	Size (kb)	Concentration, (ng/ul)	Concentration, (fmol/ul)	Volume for 50 fmol
2	pDONR-GG	3.4	100	44.6	1.1
3
4
5

Table1

2. Record the reaction setup for each reaction below. For each reaction, include:

• -- 50 fmol of each part

• -- 1.5 ul T4 ligase buffer

• -- 1.5 ul 10X BSA

• -- 1 ul BsaI

• -- 1 ul T4 ligase

• -- Water to a total volume of 15 ul.

• -- NOTE: If you're using AarI, you need to add the AarI oligo as well!

	A	B	C	D
1	A19 L4_PhlF_R1
2	pDONR-GG L1_L2	1.1 ul
3	BT-01 gBlock	1 ul
4	BT-02 gBlock	1 ul
5	Water	6.9 ul	x
6	T4 ligase buffer	1.5 ul
7	10X BSA	1.5 ul	x
8	BsaI or AarI	1 ul
9	T4 ligase	1 ul

Table2

• Set up your thermocycler

3. Program a thermocycler with the following program:

• NOTE: If you are using AarI, use 50 cycles instead of 15.

	A	B
1	Heat lid	70°
2	Start cycle	15X
3	--	37° for 1' 30"
4	--	16° for 3' 0"
5	Close Cycle
6	50° for 5'
7	80° for 10'
8	Store at 8°

Table3

• Set up your reactions

4. Fill an empty tip box with ice, put the grey platform back on, then fill with water until just under the rack.

5. Label one thin-walled PCR tube per reaction.

6. Add the reaction components. Add the water first; add the restriction enzyme and buffer last.

7. Cap the tubes. Flick a few times to mix, then pulse spin.

8. Load the tubes in the thermocycler and start the program.

• Transform

9. Transform as usual. Plate only 10 ul of the outgrowth in a 200 ul puddle of water, or you will get a lawn of colonies.