Team:Cambridge/LabBook/Week7

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Revision as of 13:46, 17 September 2010

Monday

51. Experiment: Transformation with pJS555 & pSB1C3 (Theo and Hannah)

Received a new plasmid from Jim Slock, so have transformed some chemically competent cells with it to build up a stock and test it later/we think it will glow for longer than the previous one (says Mr. Lovely)).

Theo transformed TOP10 with pSB1C3 from registry.

52. Experiment: Check for promoter+rbs+p.p.luc - obtain from the colony from transformation on page 37. (Paul)

Colony PCR

Take sample from colony with loop and put in 20μl of water to obtain DNA template.

Then prepare on isoFreeze PCR chiller in lister order:

10μl of 2xPhusion Master Mix
1μl of VR
1μl of VF2
1μl of DNA template
7μl of Nuclease-Free H20

Then PCR:

1. 98°C for 15 mins

2. 98°C for 10 secs

3. 65°C for 30 secs

4. 72°C for 1 min

5. repeat 2-4 35 times

6. 72°C for 10 mins

7. 4°C forever

Results:

Tube 1: 197.4ng/μl

Tube 2: 235.3ng/μl

Gel Electrophoresis

Tube 1: 194.7ng/μl
- 4μl of DNA
- 3μl of 6x Orange Loading Dye
- 13μl Nuclease Free H20

Tube 2: 235.3ng/μl
- 3μl of DNA
- 3μl of 6x Orange Loading Dye
- 14μl of Nuclease Free H20

53. Experiment: Extracting glycerol stocks of TOP10 with promoter+rbs+p.p.luc from registry. (Emily and Peter)

1. Take tube of glycerol stock from -80°C 2. Use loop/tooth pick (we used a loop) to streak on ampicillin resistance plate. Let it melt first, then streak.

NB NEVER let glycerol stocks thaw. They should be out and back in the -80°C freezer in 2-3mins max

3. Place in 30°C incubator overnight.

To be continued tomorrow.

54. Check for promoter+rbs+luc from p32 by luciferin injection. (Paul and Ben)

We used protocol described on p22.

3x Freezymes at -80°C, thawing at 37°C

55. Experiment: Gibson assembly of pBAD luxCDABEG in psB1C3

PCR Construct	Forward Primer	Tm	Reverse Primer	Temp	Temp to use	Template DNA	Amplifies
Prefix.Pbad.Start of C	9.prefix.f .pBadstart	53	1.luxCstart.r .pBadend	66.25	56	Pbad	pBAD
End of Pbad.C.D.Start of A	2.pBADend.f .luxCstart	51.76	4.luxAstart.r .luxDend	50.81	53	Phk555	CD
End of D.A.BStart of E	3.luxDend.f .luxAstart	54.32	6.luxEstart.r .luxBend	71.56	57	Phk555	AB
End of B.E.G.Suffix	5.luxBend.f .luxEstart	51.63	7.suffix.r ev.luxGend	56	54	Phk555	EG
End of G.Suffix.Plasmid.Prefix.Start of B	8.luxGend.f or.suffix	68.73	10.pBadstart.r .prefix	72	71	Plasmid Backbone (BBa_Jo4450)	pSB1C3

Added (on ice!) in 5 individual (A-E) PCR tubes

2x Phusion Master Mix	25µl
Forward Primer	0.25µl
Reverse Primer	0.25µl
Template DNA	2µl
Distilled water	22.5
	50µl

Programs on PCR machines

start	cycle	machine 1	machine 2	machine 3		end
98°C	98°C	56°C	53°C	71°C	72°C	72°C	10°C
30s	10s	15s	15s	15s	1.45min	7.5min	ad infinitum
		A&C	B&D	E
	Denaturation		Annealing		Elongation
			30x

Results: Machine 1 had a power fail very close to the end of the program Machine 2 had a loose lid; the tubes popped open and there was no liquid left Machine 3 had entirely melted the top of the PCR tube

The experiment was repeated, but this time all reactions (A-E) were run in Machine 2 (see p.38 for program).

Nanodrop Measurements

A	pBAD	455.6ng/µl
B	CD	339.3ng/µl
C	AB	937.4ng/µl
D	EG	165.1ng/µl
E	pSb1C3	430.8ng/µl
pA	pBAD	173.5ng/µl
pC	AB	159.7ng/µl
pE	974.6ng/µl

blanked with distilled water

Gel electrophoresis

Gel loading mixtures were made up according to:

	A	B	C	D	E	pA	pC	pE
6x orange LD (µl)	3	3	3	3	3	3	3	3
plasmid DNA (µl)	2	2	1	5	2	5	5	1
nuclease-free water (µl)	15	15	16	12	15	12	12	16

final volume 20µl

A 1% agarose E-gel was loaded as follows

Easyladder 2	A	B	C	D	E	pA	pC	pE
10µl	20	20	20	20	20	20	20	20

Bands were observed at the following lengths:

A	B	C	D	E	pA	pC	pE
1-2	3-5	2-3	2	2	1-2	2	1-2

Bands for pA, B, pC, D & pE were cut out the gel and DNA was extracted following the QIAquick Gel Extraction Kit protocol

Preparation of Gibson assembly 1.33x master mix

Taq ligase 40u/µl	50µl
5x isothermal buffer	100µl
T5 exonuclease 1u/µl	2µl
Phusion polymerase 2u/µl	6.25µl
Nuclease--free water	216.75
	375µl

Gibson Assembly Reaction added to PCR tube:

15µl	Gibson 1.33x master mix
1µl	purified pA
1µl	purified B
1µl	purified pC
1µl	purified D
1µl	purified pE
20µl

Incubated for 1h at 50C Stored at 4C overnight

	length		nanodrop after gel
pBAD	1210b	(A)	22.7ng/µl
CD	2388b	(B)	2.7ng/µl
AB	2097b	(C)	10.0ng/µl
EG	1863b	(D)	20.9ng/µl
pSB1C3	2072b	(E)	5.1ng/µl

Tuesday

Experiment: Transformation of TOP10, red strain and with Gibson assembly (pBAD, luxCDABEG, pSB1C3) (Will and Anja)

Followed protocol on p13+14. Transformed:

	with Gibson reactions
TOP10	1, 2, 3
red (BW25113 Δhns::kan)	1, 2, 3
black (GM230 hns-205::Tn10 TetR)	1, 2, 3

Plated 150µl on LB agar plates with Chl. each (9pl.)

Experiment: Repeat gel electrophoresis from p.39+40 (Will and Anja)

Followed protocol on p.39+40 (pcr products had been kept in -20°C overnight).

Results: A, C, D, pA & pC were of appropriate size. B (again 3-5kb) and E, pE (slightly lower than expected, closer to D than C) were of inappropriate size.

It was decided to run new PCR reactions for B (CD) and E (pSB1C3) following the protocol on p.38.

Nanodrop measurements: (blanked with Di H20)

B (CD) 980.7ng/µl

E (pSB1C3) 127.5ng/µl

Gel Electrophoresis:

Gel loading mixtures made up as follows:

	E	B
6x Orange LD (µl)	3	3
plasmid DNA (µl)	7	1
nuclease free H20 (µl)	10	16

A 1% Agarose (E-gel) was loaded as follows:

Easyladder 2	E	B	all other wells filled H20
10µl	20µl	20µl

@@ Line 455: / Line 455: @@
 Nanodrop measurements: (blanked with Di H20)
 B (CD)       980.7ng/µl
 E (pSB1C3)   127.5ng/µl
 ====Gel Electrophoresis:====