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	Ran iGem-ligTest in thermocycler		Ran iGem-ligTest in thermocycler
-	===July 19, 2010===
		+	===July 19, 2010===
	(in lab: A.V, H.B)<br>		(in lab: A.V, H.B)<br>
	<b>Objective:</b> Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the  biobasic protocol for purification of PCR products.		<b>Objective:</b> Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the  biobasic protocol for purification of PCR products.

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Back to Notebook
Back to Lab Work

Contents 1 July 2010 1.1 July 5/2010 1.2 July 5/2010 Evening 1.3 July 6/2010 1.4 July 8/2010 1.5 July 8/2010 - Evening 1.6 July 9/2010 1.7 July 10/2010 1.8 July 12/2010 1.9 July 12/2010 - Evening 1.10 July 13/2010 1.11 July 14/2010 1.12 July 15/2010 1.13 July 15/2010 Evening 1.14 July 16,2010 1.14.1 July 19, 2010

July 2010

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

Lane	Sample	Components (µL)
1	1kb Ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	1 - pBAD (A4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	2 - pBAD (A5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	3 - SRBS (A6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	4 - SRBS (A7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	5 - CFP Complete (A8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	6 - SRBS (A10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	7 - EYFP (B1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	8 - N term tag (B2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	9 - pSB NEYFP (B4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	10 - pSB NEYFP (B5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	11 - CFP (B6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	12 - pBAD-TetR (B10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	13 - D3	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	14 - C term (D4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
16	15 - C term (D5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
17	16 - pLacI (D6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
18	17 - NEYFP (E2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
19	18 - CEYFP (E6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
20	19 - CEYFP (E7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1	1kb ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	20 - EYFP (E8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	21 - EYFP (E9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	22 - EYFP (E10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	23 - ECFP (F1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	24 - ECFP (F2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	25 - ECFP (F3)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	26 - pBAD-TetR (F4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	27 - pBAD-TetR (F5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	28 - EYFP (G1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	29 - pSB CEYFP (G4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	30 - pBAD (1) (G6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	31 - pBAD (2) (G7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	32 - N term tag (G8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	33 - lumazine (G9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O

Ran gel at 100V for 45 minutes.

Picture to come!!!!!!!!!

July 5/2010 Evening

Objective: To over-express CFP complete in DH5α

Method:

1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm

July 6/2010

(In lab: JV, AV, HB)
Objective: To continue the over-expression of CFP complete in DH5α

Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)

Issue:

• After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.
  

Time (hours)	OD (600λ)
0	0.071
1	0.390
1.5(T0)	0.606
2.5 (T1)	1.250
3.5(T2)	3.04
4.5(T3)	2.75

Results: Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.

Objective: To determine if maxipreps were successful via restriction by NotI and running on 1% Agarose gel

Method:
1) Restriction:

Component	Volume (µL)
DNA	0.071
Buffer Orange	0.390
NotI	0.606
MilliQ H2O	1.250

Incubated for 1 hour at 37^oC. Heat killed on heat block at 80^oC for 20 mins.

2) 1% Agarose gel

Lane	Sample	Volume (µL)
1	1 kb Ladder	0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H2O
2	PET28a restricted	8 DNA + 2 Loading dye (6x)
3	PET28a	8 DNA + 2 Loading dye (6x)
4	Lumazine restricted	8 DNA + 2 Loading dye (6x)
5	Lumazine	8 DNA + 2 Loading dye (6x)
6	mms6 restricted	8 DNA + 2 Loading dye (6x)
7	mms6	8 DNA + 2 Loading dye (6x)
8	xylE restricted	8 DNA + 2 Loading dye (6x)
9	xylE	8 DNA + 2 Loading dye (6x)
10	Empty	Empty

Ran at 100V for 80 mins.

Picture to come!!!!!!!!!

More to fill in, but Anthony does not understand the stuff written in the lab book

July 8/2010

(In lab: JV, AV, HB, HS)
Objective:

Anthony does not understand the stuff written in the lab book.

July 8/2010 - Evening

(In lab: KG)
Objective:To transform TetR in pSB1A2 plasmid (BBa_C0040 - 2010 iGEM Distribution Kit Plate Well 4A) and pTetR in pSB1A2 plasmid (BBa_R0040 - 2010 iGEM Distribution Kit Plate Well 6) into DH5α.

Method:Used the Competent Cell Transformation Protocol as well as transformed pUC19 as a positive control.

Results:

Plate	Number of Colonies
50 µL TetR	0
200 µL TetR	5
50 µL pTetR	4
200 µL pTetR	34
50 µL pUC19	3
200 µL pUC19	4

July 9/2010

(In lab: JV)
Objective:To overexpress pLacI-mRBS-mms6-dT construct.

Method:Used the Overexpression Protocol.

Time (hours)	OD (600λ)
0	0.052
1	0.111
2	0.315
2.5	0.449
3(T0)	0.772
4(T1)	2.22
5(T2)	2.06
6(T3)	2.50

Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.

SDS PAGE picture!!!!!!!!!!!

July 10/2010

(In lab: JV)
Objective:To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.

Method:Run 1% Agarose gel

Lane	Sample	Components (µL)
1	dT	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
2	mms6 (1)	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
3	mms6 (2)	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
4	pBAD-TetR (1)	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
5	pBAD-TetR (2)	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
6	mRBS	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
7	pLacI	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
8	sRBS	2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
9	1 kb Ladder	0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O
10	Empty	Empty

Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.

AGAROSE GEL PICTURE

July 12/2010

(In lab: AV,HB,JV)
Objective: Maxiprep pLacI (A2) from glycerol stock.

Method: Used Maxiprep Protocol. Cell pellet weighed 1.02g.

Objective: Add dT to the end of mms6, xylE, and lumazine.

Method:
1) Restrict "Part 1" BioBricks: mms6, xylE, and lumazine with EcoRI and SpeI.

Component	Volume (µL)
pDNA	2
Red Buffer	2
EcoRI	0.25
SpeI	0.25
MilliQ H2O	15.5

2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.

Component	Volume (µL)	2.5x Volume (µL)
pDNA	2	5
Orange Buffer	2	5
EcoRI	0.25	0.625
XbaI	0.25	0.625
MilliQ H2O	15.5	38.75

3) 1% Agarose gel (1x TAE) of restricted DNA

Lane	Sample	Component (µL)
1	1 kb Ladder	0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O
2	dT	8 DNA + 2 loading dye (6x)
3	dT restricted	8 DNA + 2 loading dye (6x)
4	mms6	8 DNA + 2 loading dye (6x)
5	mms6 restricted	8 DNA + 2 loading dye (6x)
6	xylE	8 DNA + 2 loading dye (6x)
7	xylE restricted	8 DNA + 2 loading dye (6x)
8	Lumazine	8 DNA + 2 loading dye (6x)
9	Lumazine restricted	8 DNA + 2 loading dye (6x)
10	Empty	Empty

Ran at 100V for 43 minutes. Stained in EtBr for 10 minutes.
AGAROSE GEL PICTURE

4) Ligate restricted dT to the ends of the "Part 1" Biobricks.

July 12/2010 - Evening

(In lab: KG,TF)
Objective: Continue with addition of dT to the ends of mms6, xylE, and lumazine.

Method:

4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
dT to mms6:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
mms6	9.2
MilliQ H2O	0.25

dT to xylE:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
xylE	3.4
MilliQ H2O	6.05

dT to lumazine:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
lumazine	3.35
MilliQ H2O	6.1

Ligations were incubated at room temperature overnight.

Objective: Prepare mastermix for four PCR reactions for following day.

Method:

Component	Volume (µL)	5x Volume (µL)
10mM dNTPs	1	5
5x Phusion Buffer	4	20
Forward Primer (VF2)	1	5
Reverse Primer (VR)	1	5
MilliQ H2O	10.8	54

July 13/2010

(in lab: JV)
Objective: To determine if ligations of previous day (July 12/2010) were successful.
Method:
1) Use prepared mastermix to run a PCR of ligated mms6-dT, ligated xylE-dT, ligated lumazine-dT, and control-dT
- To each PCR tube, add 17.8&mirco;L of mastermix, 0.2µL of Phusion DNA polymerase, and 2µL of template DNA.
- Ran PCR's for 36 cycles using the iGEM preset.

2) 2% Agarose gel

Lane	Sample	Components (µL)
1	50 bp Ladder	1 ladder + 2 loading dye (6x) + 7 MilliQ H2O
2	mms6-dT	8 PCR product + 2 loading dye (6x)
3	xylE-dT	8 PCR product + 2 loading dye (6x)
4	lumazine-dT	8 PCR product + 2 loading dye (6x)
5	dT	8 PCR product + 2 loading dye (6x)
6	Empty	Empty
7	Empty	Empty
8	Empty	Empty
9	Empty	Empty
10	Empty	Empty

Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.

AGAROSE GEL PICTURE

July 14/2010

(in lab:J.S, K.G )
Objective: Inoculate culture for maxi prep with placI

Method: To a 450mL solution of LB media 4.5µL of ampicillin was added with glycerol placI aseptically.

---

(in lab:J.S, K.G )
Objective: Restriction of dT and mms6

protocol

1)

Component	Volume (µL)
Buffer&*	2
10x T4 Milli Q H2O	15.5
pDNA**	2
Restriction enzymes***	0.25

* Orange buffer was used for dT, and Red buffer was used for mms6

**pDNA was dT and mms6

***Restriction enzymes for dT were XbalI and EcoRI, and for mms6 SpeI and EcoRI

2) Reaction was incubated at 37⁰C for 1 hour. Start 8:50pm till 9:50pm After incubation reaction was heat shocked at 80⁰C for 20 minutes

---

(in lab:J.S, K.G )
Objective: Ligation of dT to each of mms6, xylE and lumazine.

protocol

1)

dT to mms6:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
mms6	9.2
MilliQ H2O	0.25

dT to xylE:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
xylE	3.4
MilliQ H2O	6.05

dT to lumazine:

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
lumazine	3.35
MilliQ H2O	6.1

2) Incubated reaction overnight at room temperature

July 15/2010

(in lab: AV)
Objective: Maxiprep pLacI and mms6.

component	pallet weight (g)
mms6	1.02
pLacI	1.54

July 15/2010 Evening

(in lab: AV)
Objective: transform ligations from July 14 and July 12,2010;xylE/dt, mms6/dt lumazine/dt into DH5&alpha. Also to transform mms6 from July 6 and July 10 into Bl21(DE3)

Protocol: to transform competent cells see protocol:[1]

* cells were incubated on ice for 30 minutes started at 7:00-7:30. **incubated for 1 hour from 8:00 till 9:00

Results
plate	# of colonies	Components (µL)
1	200µL xylE-dt July 12	0
2	200µL mms6-dt July 12	1
3	200µL lumazine-dt	1
4	200µL mms6 maxiprep July 6	Lawn
5	200µL positve control puC19 into BL21(DE3)	Lawn
6	200µL xylE-dt July 14	0
7	200µL mms6-dt July 14	120
8	200µL lumazine-dt July14	0
9	200µL mms6 maxiprep July 10	.

*because of a shortage of plates not all transformations were plated at 50µL and 200µL

July 16,2010

(in lab: M.C, D.M)
Objective: PCR amplify mms6-dT, xylE-dT, lumazine-dT legations along with dT maxi prep for comparison

Protocol:

Master Mix

Component	Volume (µL)
Milli-Q H2O<	54
Pfu Buffer + MgSO4	20
10 mM dMTPs	5
forward primer	5
reverse primer	5

89 µL TOTAL--> 17.8 into each PCR reaction

+ 2 µL ligation + 2 µL Pfu polymerase

Ran iGem-ligTest in thermocycler

July 19, 2010

(in lab: A.V, H.B)
Objective: Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the biobasic protocol for purification of PCR products.

Objective: Add pLacI to sRBS and add dT to xylE and lumazine

Method:

Restrict pLacI, xylE and lumazine with SpeI and EcoRI and restrict dT and sRBS with EcorI and XbaI

1)Restrictions:

pLacI, xylE, and Lumazine

Component	Volume (µL)
Milli-Q H2O	15.5
Red Buffer4	2
SpeI	0.25
EcoRI	0.25
pDNA	2

dT

Component	Volume (µL)
Milli-Q H2O	38.75
Orange Buffer4	5
XbaI	0.625
EcoRI	0.625
pDNA	5

sRBS

Component	Volume (µL)
Milli-Q H2O	15.5
Red Buffer4	2
xBal	0.25
EcoRI	0.25
pDNA	2

Restriction incubation at 37.5⁰C started at 11:55am. Ended at 12:55pm Heat killed enzymes at 80⁰C for 20 minutes

2)Restrictions were run on a 1% agarose gel (1 X TAE) 1% Agarose gel

Lane	Sample	Components (µL)
1	1kB Ladder	0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H2O
2	pLacI	6 pDNA + 2 loading dye (5x)
3	pLacI restriction digest	6 pDNA + 2 loading dye (5x)
4	sRBS	6 pDNA + 2 loading dye (5x)
5	sRBS restriction digest	6 pDNA + 2 loading dye (5x)
6	xylE	6 pDNA + 2 loading dye (5x)
7	xylE restriction digest	6 pDNA + 2 loading dye (5x)
8	lumazine	6 pDNA + 2 loading dye (5x)
9	lumazine restriction digest	6 pDNA + 2 loading dye (5x)
10	dT	6 pDNA + 2 loading dye (5x)
11	dT restriction digest	6 pDNA + 2 loading dye (5x)

Gel ran for 70 minutes at 100V and was stained in EtBr for 10 minutes

AGAROSE GEL PICTURE

Results after quantifying restriction digest

Lane	Content	Quantity of DNA (ng/µL)
1	1kB Ladder	12.5
3	pLacI restriction digest	5.21
5	sRBS restriction digest	3.72
7	xylErestriction digest	3.36
9	Lumazine restriction digest	2.63
11	dT restriction digest	2.98