Team:MIT/miRNA Group Notebook pDEST mCherry

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PCR diary for pDEST mCherry with GG cassettes 2.0

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-08-24 to 2016-10-09

Wednesday, 8/24

Starting Over, Primers

We realized the golden gate wouldn't work so we got new primers

	A	B	C
1		08/12/16 Sent to Brian to order
2	#	Name	Primer Sequence (5'-3') - capitalize flap
3	55	iGEM 55 DONR-F-2.0	CCCGTGACTAGTGCTTATGTCTTCaattcacctgctgacaggttgag
4	56	iGEM 56 DONR-R-2.0	CTCGGTATCGATGTTGATGTCTTCcacctgcactgaagctgag

Table1

\

Primers only designed for restriction digest + ligation

PCR

Redoing for all -- even though DC RE should still be fine..

	A	B	C	D
1	Nickname	Template	Forward Primer	Reverse Primer
2	DC	pDest_mCherry	iGEM 17 pDEST_mCherry Restriction Forward	iGEM 18 pDEST_mCherry Restriction Reverse
3	GG	pDONR_GG (L4, R1)	iGEM 55 DONR-F-2.0	iGEM 56 DONR-F-2.0

Table20

Prepare on Ice in this order

	A	B
1		Volume per rxn
2	Ultrapure H2O	20
3	Template (dil to 2 ng/uL)
4	Fwd Primer (10 uM)	2.5
5	Rev Primer (10 uM)	2.5
6	Q5 Master Mix (2X)	25
7	Total	30
8

Table2

Protocol: Q5 repressors b

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	68	30	DC RE
6		67	30	GG RE
7	Extend	72	90
8	Cycle Ends
9	Final Extension	72	120
10	Store	4	forever

Table21

Thursday, 8/25

Gel Extraction

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

	A	B	C
1	Lane	Nickname	Expected Length
2	1	NEB Ladder 1 kb	--
3	2	DC	3836bp
4	3	DC	3836bp
5	4	skip
6	5	NEB Ladder 1 kb	--
7	6	GG RE	751bp
8	7	GG RE	751bp

Table22

Used wrong primers so i didn't get any DC :P can try to move forward with DC RE from before (should be fine).

	A	B	C	D	E	F
1	Tube	Weight (mg)	QG Added (uL)	Isopropanol Added (uL)	Nanodrop (ng/uL)
2	DC RE	286	1716	286	32.9	Done 7/28, in diary 1.0
3	GG 1	128	768	128
4	GG2	143	858	143

Table23

Digestion (10 uL)

	A	B
1	Name on Tube	Concentration (ng/uL)
2	DC RE	32.9
3	GG RE 1	53.1

Table24

	A	B	C	D	E
1		"D+G RE"	Neg
2	DC RE DNA (100 ng)	3 uL	3
3	GG RE DNA (100 ng)	2 uL	0
4	10X Cut Smart	1 uL	1
5	SpeI	1 uL	1
6	ClaI	1 uL	1
7	Nuclease free water	2 uL	4
8	total volume	10 uL	10 uL

Table25

Digested for 1.5 hours at 37C

Heat inactivated for 40 mins at 80C (should have only been 20 mins.. ops)

Friday, 8/26

Ligation

Ligating: D+G as well as negative control (D alone)

	A	B	C
1		"xp"	"t4"
2	Buffer	1 uL	1 uL
3	DNA	4 uL mixture	4 uL mixture
4	Insert	N/A	N/A
5	H2O	4 uL	4 uL
6	Ligase	1 uL express	1 uL t4

Table27

Gently mix the reaction by pipetting up and down, and microfuge briefly.

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.

Transformation

	A	B	C	D
1	Label	What's in it	Amount (uL)
2	D+G XP	pDest_mCherry + GG with XP ligase	5
3	Neg XP	Dest alone with T4 ligase	5
4	D+G T4	pDest_mCherry + GG	5	Saving these
5	Neg T4	Dest alone	5
6	pUC 19 (1 pg/ul)	control	1
7
8
9

Table3

Plating with beads, 5 ul outgrowth + 200 ul water for some or 100 ul of outgrowth (plate labelled)

Saturday, 8/27

20160827_133826.jpg

20160827_133823.jpg

20160827_133817.jpg

Sunday, 8/28

Picked overnights and cultured in 4-5 ml of LB amp

Picked:

3 blue colonies

1 that looked mixed / both colors involved

1 red

Tuesday, 8/30

Overnights looked like this the next day:

20160828_095813.jpg

20160828_095849.jpg

20160828_095922.jpg

Pellets looked like this:

20160828_102316.jpg

20160828_102355.jpg

Miniprep Results

Lost Blue 1 :( RIP

	A	B
1	Name on Tube	Concentration (ng/uL)
2	blue 2	459.7
3	blue 3	370.2
4	both	279.1
5	red	291.6

Table4

Confirmation Digest

Digest with BamHI, NcoI

	A	B	C	D	E	F	G	H
1		Uncut (B2)	bamHI (B2)	ncoI (B2)	B2	B3	Both	Red
2	DNA (~120 ng/ul)	3	3	3	3	3	3	3
3	10x NEB 3.1	1	1	1	1	1	1	1
4	BamHI	0	1	0	1	1	1	1
5	NcoI	0	0	1	1	1	1	1
6	Nuclease free water	6	5	5	4	4	4	4
7	total volume	10	10	10	10	10	10	10

Table5

Digested for 1 hours at 37C

Not enough NcoI to do all digests

Confirmation Gel

repmcherrygggel.JPG

weisslab 2016-08-28 14hr 26min.jpg

B2 and B3 look good!

Sunday, 10/9

Diagnostic Digest

Confirming:

Insertion of promoter/gene

presence of LacZ

CF 10/6 LR Code:

1: pBM3R1: 2kturn - TP901

2: pERE3: TP901

3: pERE5: TP901

4: pERE3: mKate

5: pERE5: mKate

6: pHybrid: BM3R1

(Used the 1 version for everything)

ONLY used "1" version for all

2: pERE3: TP901 3: pERE5: TP901

	A	B	C	D	E	F	G	H	I
1		2 Uncut	2 Single (2B)	2 Single (2N)	2 Double	3 Double
2	DNA (~100 ng/ul)	3	3		3	3
3	10X Cut Smart	1	1	1	1	1
4	BamHI	0	0	1	1	1
5	NcoI	0	1	0	1	1
6	Nuclease free water	6	5	8	4	4
7	total volume	10	10	10	10	10

Table6

4: pERE3:mKate 5: pERE5:mKate

	A	B	C	D	E	F	G	H	I
1		4 Uncut	4 Single	4 Single	4 Double	5 Double
2	DNA (~100 ng/ul)	3	3	3	3	3
3	10X Cut Smart	1	1	1	1	1
4	StuI	0	0	1	1	1
5	NcoI	0	1	0	1	1
6	Nuclease free water	6	5	5	4	4
7	total volume	10	10	10	10	10

Table7

Gel

Ran in the above order

clipboard_2016-10-09_15:33:26.png

weisslab 2016-10-09 15hr 46min.jpg

ERE5-TP901-GG-1 (3-1) andERE5-mKate-GG-1 (5-1) seem good

will attempt other golden gates with ERE3-TP901-GG-2 (2-2) and ERE-3-mKate-GG-2 (2-4)

PCR Diary for pDEST_mCherry and GG cassettes (06/27/16 - 8/24)

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-06-27 to 2016-08-10

Monday, 6/27

Ran 4 PCR reactions

Set Up:

	A	B	C	D
1	Nickname	Template	Forward Primer	Reverse Primer
2	DEST	pDEST-T	iGEM 09 pDEST-T R4_GTW6_R2 Forward	iGEM 10 pDEST-T R4_GTW6_R2 Reverse
3	RED	pL2f1 091	iGEM 11 pLacIq_mCherry Forward	iGEM 12 pLacIq_mCherry Reverse
4	GG cut	pDONR-GG L4_LacZa_L1	iGEM 19 pDONR_GG L4_LacZa_R1 Restriction Forward	iGEM 20 pDONR_GG L4_LacZa_R1 Restriction Forward
5	GG Gibson	pDONR-GG L4_LacZa_L1	iGEM 15 pDONR_GG L4_LacZa_R1 Gibson Forward	iGEM 16 pDONR_GG L4_LacZa_R1 Gibson Reverse

Table1

Protocol: "Q5 repressors1"

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	67	30	DEST
6		70	30	Red
7		72	30	GG cut and GG gibson
8	Extend	72	90
9	Cycle Ends
10	Final Extension	72	120
11	Store	4	forever

Table3

Tuesday, 6/28

Ran a Diagnostic Gel

	A	B	C
1	Lane	Nickname	Expected Length
2	1	Bioline Ladder 1 kb	--
3	2	Bioline Ladder 1 kb	--
4	3	DEST	2972
5	4	RED	878
6	5	GG cut	738
7	6	GG Gibson	749

Table2

06-28-16 pcr products.jpg

Wednesday, 6/29

Checked concentration after PCR Purification

	A	B	C
1	Name on Tube	Concentration (ng/uL)
2	DEST PCR	23.6
3	mCherry PCR (formerly known as RED)	28.3
4	GG cut PCR	13.1
5	GG gib PCR	23.9

Table4

Stored in -20C Repressor Box

Did Double Digest Protocol (In 10 uL total)

From NEB http://nebcloner.neb.com/#!/

Adjusting the NEB protocol per Brian's recommendations

	A	B	C
1		DEST	mCherry
2	DNA (100 ng)	4 uL	3.5 uL
3	10X Cut Smart	1 uL	1 uL
4	AsiSI	1 uL	1 uL
5	XhoI	1 uL	1 uL
6	Nuclease free water	3 uL	3.5 uL

Table5

Positive Control for transformation:

	A	B
1		pUC19 nondilute
2	DNA (100 ng)	2 uL
3	10X cut smart	1 uL
4	SphI	1 uL
5	Water to 10 uL	6 uL
6

Table6

Incubate all reactions at 37C per NEB protocol (1 hour)

Heat inactivated at 80C for 20 mins per NEB protocol.

Holding off on digesting GG cut and GG gibson until pDEST_mCherry is linearized

Stored in -20C Repressor Box

Ligation in 10 uL Reaction

Also used NEB T4 Ligase protocol for sticky ends + transformation into chemically competent cells. Changed amounts according to table below.

Want reaction to be 1:3 (backbone:insert).

Backbone = DEST

insert = mCherry

Note: T4 buffer is in aliquots in our -20C in a tip box. T4 Ligase is in enzyme box. Remove T4 Ligase only when ready to add to reaction

	A	B	C	D
1		Tube: Dest +Cherry	Tube: DEST alone	Tube: PUC alone
2	Buffer	1 uL	1 uL	1 uL
3	Backbone	2 uL	2 uL	2 uL
4	Insert	2 uL	N/A	N/A
5	H2O	4 uL	6 uL	6 uL
6	T4 Ligase	1 uL	1 uL	1 uL

Table7

Stored in -20C Repressor Box

Transformation

Sample: DEST + mCherry

Negative Control: Ligation with backbone only (no insert)

Positive Control: pUC19 cut once and religated.

Positive Control: pUC19 never cut

Stored at 4C after incubation

Thursday, 6/30

Results of plating:

Controls fine, Sample only has one colony. Leaving sample at room temp to see if colony turns red.

Also retransforming ligation today.

Nothing turned red -->

Alot of plating and stuff happened that was kinda recorded in the experiment 1 folder Digest and Gel

Thursday, 7/7

REDO Double Digest Protocol (In 20 uL or 10 uL total)

From NEB http://nebcloner.neb.com/#!/

Brian suggested

	A	B
1		One Pot
2	DEST DNA (100 ng)	4 uL
3	mCherry DNA (100 ng)	3.5 uL
4	10X Cut Smart	2 uL
5	AsiSI	1 uL
6	XhoI	1 uL
7	Nuclease free water	8.5 uL

Table8

	A	B	C
1		DEST	mCherry
2	DNA (100 ng)	4 uL	3.5 uL
3	10X Cut Smart	1 uL	1 uL
4	AsiSI	1 uL	1 uL
5	XhoI	1 uL	1 uL
6	Nuclease free water	3 uL	3.5 uL

Table9

Incubate at 37C for 3 hours

Redo Ligation in 10 uL Reaction

Also used NEB T4 Ligase protocol for sticky ends + transformation into chemically competent cells. Changed amounts according to table below.

Want reaction to be 1:3 (backbone:insert).

Backbone = DEST

insert = mCherry

Note: T4 buffer is in aliquots in our -20C in a tip box. T4 Ligase is in enzyme box. Remove T4 Ligase only when ready to add to reaction

	A	B	C	D
1		Tube: Dest + mCherry	Tube: DEST alone	Tube: One Pot
2	Buffer	1 uL	1 uL	1 uL
3	Backbone	2 uL	2 uL	4 uL mixture
4	Insert	2 uL	N/A	N/A
5	H2O	4 uL	6 uL	4 uL
6	T4 Ligase	1 uL	1 uL	1 uL

Table10

Gently mix the reaction by pipetting up and down, and microfuge briefly.

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.

Plating results: Very few colonies throughout, trashed

Friday, 7/8

Redid PCR reactions

per Brian's tube set up protocol

Set Up:

	A	B	C	D
1	Nickname	Template	Forward Primer	Reverse Primer
2	DEST	pDEST-T	iGEM 09 pDEST-T R4_GTW6_R2 Forward	iGEM 10 pDEST-T R4_GTW6_R2 Reverse
3	RED	pL2f1 092	iGEM 11 pLacIq_mCherry Forward	iGEM 12 pLacIq_mCherry Reverse
4	GG cut	pDONR-GG L4_LacZa_L1	iGEM 19 pDONR_GG L4_LacZa_R1 Restriction Forward	iGEM 20 pDONR_GG L4_LacZa_R1 Restriction Forward
5	GG Gibson	pDONR-GG L4_LacZa_L1	iGEM 15 pDONR_GG L4_LacZa_R1 Gibson Forward	iGEM 16 pDONR_GG L4_LacZa_R1 Gibson Reverse

Table11

Protocol: "Q5 repressors1"

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	67	30	DEST
6		70	30	Red
7		72	30	GG cut and GG gibson
8	Extend	72	90
9	Cycle Ends
10	Final Extension	72	120
11	Store	4	forever

Table12

	A	B	C	D	E	F
1	Top Lane	Nickname	Expected Length		Top Lane	Nickname
2	1	Bioline Ladder 1 kb	--		1	blank
3	2	1 kb Ladder	--		2	1 kb ladder
4	3	DEST	2972		3	Trinh 1
5	4	RED	878		4	Trinh 2
6	5	GG cut	738
7	6	GG Gibson	749

Table13

Protocol: Q5 repressors 1

Ran on gel, stored bands at 4C

Monday, 7/11

	A	B	C	D	E
1	Tube	Weight (mg)	QG Added (uL)	Isopropanol Added (uL)
2	DEST	154	924	154
3	RED	160	960	160
4	GG gib	283	1698	283
5	GG cut	210	1260	210

Table14

REDO Double Digest Protocol (In 20 uL or 10 uL total)

From NEB http://nebcloner.neb.com/#!/

Brian suggested

	A	B	C
1	Name on Tube	Concentration (ng/uL)
2	DEST PCR	10.8
3	mCherry PCR (formerly known as RED)	40.7
4	GG cut PCR	44
5	GG gib PCR	45.4

Table17

	A	B
1		One Pot
2	DEST DNA (100 ng)	10 uL
3	mCherry DNA (100 ng)	2.5 uL
4	10X Cut Smart	2 uL
5	AsiSI	1 uL
6	XhoI	1 uL
7	Nuclease free water	3.5 uL
8	total volume	20 uL

Table15

	A	B	C
1		DEST	mCherry
2	DNA (100 ng)	10 uL	2.5 uL
3	10X Cut Smart	1.5 uL	1 uL
4	AsiSI	1 uL	1 uL
5	XhoI	1 uL	1 uL
6	Nuclease free water	1.5 uL	4.5 uL
7	total volume	15 uL	10 uL

Table16

Incubated at 37C for 3 hours

Wednesday, 7/13

Redoing Ligation

blue = brian's ligation mixes

green = standard

D = pDest

DM = pDest mCherry (1 pot)

+ = pDest + mCherry

Redo Transformation

per Brian's benchling protocol --> with 5 uL of DNA added instead of 2 uL

Wednesday, 7/20

For the previous diagnostic gel digest (and sequencing) results of pDEST+mCherry, please refer to this note.

Second Diagnostic Gel Digest

Enzymes used for running the pDest+mCherry miniprep products on gel: Nco1 (single cut), Xho1 (single cut)

ALL MEASUREMENTS BELOW ARE ul.

	A	B	C	D	E	F	G	H	I	J	K	L	M	N	O	P	Q
1	SAMPLE LABEL	LADDER	1	2	3	4	5	6	LADDER	7	8	9	10	11	12	LADDER	SAMPLE LABEL
2	PLASMID		pD+mC 1	pD+mC 1	pD+mC 1	pD+mC 1	pD+mC 2	pD+mC 3		pDmC 1	pDmC 1	pDmC 1	pDmC 1	pDmC 2	pDmC 3		PLASMID
3	H2O		6	5	5	4	4	4		6	5	5	4	4	4		H2O
4	BUFFER		1	1	1	1	1	1		1	1	1	1	1	1		BUFFER
5	DNA		3	3	3	3	3	3		3	3	3	3	3	3		DNA
6	ENZYME 1 (Nco1)		N/A	1 ul	N/A	1 ul	1 ul	1 ul		N/A	N/A	1 ul	1	1	1		(Nco1) ENZYME 1
7	ENZYME 2 (Xho1)		N/A	N/A	1 ul	1 ul	1 ul	1 ul		N/A	1 ul	N/A	1	1	1		(Xho1) ENZYME 2
8	WELL NUMBER	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	WELL NUMBER

Table18

why do we have 2 separate gels for the samples on the table above?

during the loading of the gel, due to a confusion we skipped 1 well between Sample 8 and Sample 9.

	A	B	C	D	E	F	G	H	I	J	K	L	M	N	O	P	Q
1	well number	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16
2	sample label (IDEAL)	LADDER	1	2	3	4	5	6	LADDER	7	8	9	10	11	12	LADDER	NO NEED
3	sample label (1st gel)	LADDER	1	2	3	4	5	6	LADDER+7	8	9	SKIP	9	10	11	12	LADDER
4	sample label (2nd gel)	SKIP	SKIP	LADDER	1	2	3	4	5	6	LADDER	7	8	10	11	LADDER

Table19

GEL 1

pdest-mcherry_16072_gel1_manualExposure.jpg

annotated_pdest-mcherry_160720_gel1_manualExposure.jpg

GEL 2

pdest-mcherry_16072_gel2_manualExposure2.jpg

annotated_pdest-mcherry_160720_gel2_manualExposure2.jpg

Monday, 7/25

Look at testing pDEST_mCherry to see optimal conditions for use.

Results: pDest_mCherry was successful in Tal21:EYFP LR reactions. Both the pDest + mCherry colony 1 and pDest_mCherry_one_pot colony 1 were used. Different of transformed cells were plated: 100uL of transformation, 20ul transformation/200uL water, and 10uL transformation/200 uL water.

The easiest plate to pick colonies was the 10uL/200uL plate using pDest + mCherry. Please see "Testing pDest mCherry (Making Tal21:EYFP)"

Thursday, 7/28

PCR Amplification

PCR for pDest_mCherry (previously referred to as pDest + mCherry colony 1 or 2) backbone, PCR Golden Gate Cassette to include new Q sites and either restricition enzyme sites or Gibson complementary sequence

	A	B	C	D
1	Nickname	Template	Forward Primer	Reverse Primer
2	DC RE	pDest_mCherry	iGEM 17 pDEST_mCherry Restriction Forward	iGEM 18 pDEST_mCherry Restriction Reverse
3	DC Gib	pDest_mCherry	iGEM 13 pDEST_mCherry Gibson Forward	iGEM 14 pDEST_mCherry Gibson Reverse
4	GG RE	pDONR_GG	iGEM 39 pDONR-GG L4_LacZa_R1_RE_F	iGEM 40 pDONR-GG L4_LacZa_R1_RE_R
5	GG Gib	pDONR_GG	iGEM 37 pDONR-GG L4_LacZa_R1_Gib_F	iGEM 38 pDONR-GG L4_LacZa_R1_Gib_R

Table20

Prepare on Ice in this order

9uL Ultrapure Water

1uL Template DNA (Any Concentration less than 1 ug)-nanodrop verification.

1.25uL 10uM Forward Primer

1.25uL 10uM Reverse Primer

12.5uL 2X Q5 Master Mix

	A	B	C	D
1	Step	Temperature (C)	Time (s)	Sample
2	Initial Denaturation	98	30
3	Cycle 30 x
4	Denature	98	10
5	Anneal	68	30	DC RE, DC Gib
6		67	30	GG RE, GG Gib
7		58	30	miR9, miR135
8	Extend	72	90
9	Cycle Ends
10	Final Extension	72	120
11	Store	4	forever

Table21

Gel Extraction

	A	B	C
1	Lane	Nickname	Expected Length
2	1
3	2	Bioline Ladder 1 kb	--
4	3	Recombinse Group	--
5	4	DC RE	3836bp
6	5	DC Gib	3812bp
7	6	GG RE	751bp
8	7	GG Gib	774bp

Table22

	A	B	C	D	E
1	Tube	Weight (mg)	QG Added (uL)	Isopropanol Added (uL)	Nanodrop (ng/uL)
2	DC RE	286	1716	286	32.9
3	DC Gib	239	1434	239	27.5
4	GG RE	329	1974	329	52.5
5	GG Gib	211	1266	211	40.5

Table23

Monday, 8/1

Digestion (10 uL rxn)

From NEB http://nebcloner.neb.com/#!/

Brian suggested

	A	B
1	Name on Tube	Concentration (ng/uL)
2	DC RE	32.9
3	GG RE	52.5

Table24

	A	B
1		"D+G RE"
2	DC RE DNA (100 ng)	3 uL
3	GG RE DNA (100 ng)	2 uL
4	10X Cut Smart	1 uL
5	SpeI	1 uL
6	ClaI	1 uL
7	Nuclease free water	2 uL
8	total volume	10 uL

Table25

Incubate at 37C for ~1.5 hr

	A	B	C
1		"xp"	"t4"
2	Buffer	1 uL	1 uL
3	DNA	4 uL mixture	4 uL mixture
4	Insert	N/A	N/A
5	H2O	4 uL	4 uL
6	Ligase	1 uL express	1 uL t4

Table27

Gently mix the reaction by pipetting up and down, and microfuge briefly.

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.

Calculating for Gibson Assembly

Calculations

x = [(base pairs)(650)(0.2) ] / [(1,000)(nanodrop count)]. If size is <200 bases, multiply x by 5 (see source beneath).

	A	B	C	D	E	F
1		ng/uL	Dilution	base pairs	fmol in rxn	uL in rxn
2	DC gib	27.5	27.5	3812	40	3.60
3	GG gib	40.5	20	774	40	1.01

Table26

Up rxn to 40 uL???? --> Ask brian tomorrow -----> Don't

Tuesday, 8/2

Suggestions from Brian

To increase yield of gel extraction: up total pcr volume to 50 uL

Adjusting Gibson: his protol calls for 100-200 fmol. In reality, can scale reaction to as little as 7 fmol.

Try to keep Gibson total volume 10 ul, with up to 5 uL for DNA addition. keep DNA equimolar.

Gibson Assembly

	A	B	C
1		"Gib"	"Ctrl"
2	Water	0.4	3
3	Master Mix	5	5
4	DC gib	3.6	1
5	GG gib	1	1

Table28

Tuesday, 8/9

Diagnostic Gel:

	A	B	C	D	E
1	NEB quick load 2 log ladder		Nanodrop:
2	uncut
3	bamhI
4	NcoI
5	T4 A
6	T4 B	winner!!!!	146.4 ng/ul
7	XP A
8	XP B
9	Red A
10	Red B

Table29

repmcherrygggel.JPG

weisslab 2016-08-09 18hr 34min.jpg

Wednesday, 8/10

Golden Gate Assembly

To do::

Generate pDest-mCherry-GG construct on venchling

Secure bbsI enzyme

Testing pDEST mCherry (Making Tal21:EYFP)

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Dates: 2016-07-21 to 2016-07-26

Thursday, 7/21

All DEST's diluted to ~10 fmol/ul

	A	B	C	D	E
1	Name on Tube	D	MD	+
2	DEST	"DEST-T"	"MD"	"+"
3	ENTR L4-R1	4xTal21	4xTal21	4xTal21
4	ENTR L1-L2	eyfp	eyfp	eyfp
5

Table1

Friday, 7/22

Transformed and plated.

Transformeed as usual, plated:

- 10 uL of transformation + 200 uL of water

- 20 uL of transformation + 200 uL of water

Monday, 7/25

Results of plating

20160723_154331.jpg

20160723_154342.jpg

20160723_154348.jpg

20160723_154352.jpg

20160723_154402.jpg

20160723_154413.jpg

20160723_154421.jpg

20160723_154430.jpg

20160723_154442.jpg

20160723_154450.jpg

Tuesday, 7/26

nothing

nothing

uncut

pst1

ecor1

ladder

pdest

a

b

nothing

nothing

nothing

weisslab 2016-07-25 18hr 48min.jpg

Testing pDEST_mChery_GG

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-09-23

Friday, 9/23

Suggested Approach

These are just some notes to help you get started.

*Our pDEST_mCherry_GG 2.0 has both an mCherry gene and a LacZ cassette. This means colonies may appear both red and blue. From my experience, the colonies containing mCherry AND LacZ look just straight up blue.

Thus, In order to test pDest_mCherry_GG, I would suggest the following steps

1.

Do an LR, plating on LB-amp plates (no xgal so colonies won't be blue), and then select for the non-red colonies FIRST. This will give you the colonies with the promoter and gene inserted that still have the LacZ cassette.

Destination: pDEST_mCherry_GG_2.0 (Sequence)

Location: -20C, pink "iGEM Repressor + miRNA..." Box, in minipreps section. Label: DEST + GG B2 or B3 (choose either)

Promoter: hef1a

Location: -20C, working stocks box

Gene: choose between bfp, mkate, eyfp

Location: -20C, working stocks box

2.

Grow up overnight cultures then miniprep. Save a little of the culture (1-2ml for later)

3.

Design and run a gel digest & sequence to confirm gene and promoter have been inserted.

4.

Once confirmed, do golden gate with that tube. Follow protocol Golden Gate Protocol

5.

Mini and midiprep

6.

Design and run a restriction digest to ensure 451a sensor has been inserted. Could also try to reconfirm gene and promoter are also there.

Materials for Golden Gate

Follow materials listed in Golden Gate Protocol. These are just notes for a few of the materials/parts of the procedure.

●

"Backbone plasmid" --> Instead of using pDONR, we want to use miniprepped LR product that was created (as stated above)

Location: This is what you miniprepped and confirmed. You choose where to put it, but you should store it in our pink box in -20C, in miniprep section with distinct label.

●

Parts being assembled --> Choose the ultramer for 451 a . This is the only part necessary.

Location: -20C, "Glycerol + siRNA + ultramers box", Labelled 451a ultra (concentration is low, 17.2 ng/ul, you may need to calculate how much you need for golden gate early on and pcr amplify more if necessary.

**Don't leave this box out on the benchtop***