Team:Stockholm/15 September 2010

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Latest revision as of 11:03, 26 October 2010

Andreas

Assembly of new parts

Transformation results

Good (white) colony yield on all plates. Picked 4 from each for colony PCR.

Colony PCR

pSB1K3.N-TAT⋅SOD⋅His: TAT.SH 1-4
pSB1K3.N-Tra10⋅SOD⋅His: Tra10 SH 1-4
pSB1A2.RBS.yCCS: RBS.y 1-4
pEX.SOD 1-4

Controls

pEX.RFP
pSB1A2.RBS
pSB1C3.SOD
pEX.SOD⋅His
pSB1C3.SOD⋅His

Standard colony PCR settings

Elongation: 1:20

Gel verification

Gel 1

Colony PCR gel verification of N-TAT⋅SOD⋅His and N-Tra10⋅SOD⋅His assemblies in pSB1K3.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder.

1 % agarose, 90 V

Expected bands

pSB1K3.N-TAT⋅SOD⋅His: 854 bp
pSB1K3.N-Tra10⋅SOD⋅His: 884 bp
pSB1C3.SOD⋅His: 815 bp

Results
Very unclear gel. Ladders are impossible to read. Two N-Tra10 clones (Tra10 SH1 and 2) seem to have migrated slightly slower than the SOD⋅His control, which may be a sign of successful insertion of Tra10. PCR samples will be analyzed again tomorrow.

Gel 2

Colony PCR gel verification of RBS(BBa_B0034).yCCS assemblies in pSB1A2.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder

1 % agarose, 90 V

Expected bands

pSB1A2.RBS.yCCS: 1018 bp
pSB1A2.RBS: 250 bp

Results
All bands at 250 bp, clearly showing that insertion of yCCS was unsuccessful. Incomplete digestion of pSB1A2.RBS with SpeI and PstI might be the cause of problem.

Gel 3

Colony PCR gel verification of pEX.SOD.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder

1 % agarose, 90 V

Expected bands

pEX.SOD: 678 bp
pEX.RFP: 1385 bp
pEX.SOD⋅His: 702 bp

Results
No pEX.SOD bands whatsoever, probably due to lack of template in PCR tubes. Will re-run colony PCR tomorrow.

Plasmid prep

From Johan's ON culture

DNA concentration
Sample	Conc [ng/μl]	A₂₆₀/A₂₈₀
pSB1C3.C-TAT	123.7	1.82

Assembly of His⋅SOD⋅C-TAT

Digestion

[pSB1C3.C-TAT] = 123.7 ng/μl

10X FastDigest buffer	3
dH₂O	8.8
DNA (2 μg)	16.2
NgoMIV	1
FD PstI	1
	30 μl

Incubation: 37 °C, 2:00 (NgoMIV); 0:30 (FD PstI)
Inactivation: 80 °C, 20 min

Ligation

Vector: [Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/μl
Insert 1: [Dig pMA.His⋅SOD E+A 14/9] = 66.6 ng/μl
Insert 2: [Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/μl

10X T4 Ligase buffer	2
dH₂O	0
Vector DNA	1.5
Insert 1 DNA	4.5
Insert 2 DNA	11
T4 DNA ligase	1
	20 μl

Incubation: 22 °C, 15 min

Transformation

Standard transformation

1 μl ligation mix
Km 50 plate

ON cultures

3 ml LB + Cm 25; 30 °C
- pSB1C3.C-TAT
5 ml LB + Cm 25; 37 °C, 225 rpm
- pSB1C3.N-TAT
- pSB1C3.N-Tra10
- pSB1C3.SOD

Mimmi

SOD.his / his.SOD / yCCS

glycerol stock / over expression

	glycerol stock	over expression
SOD.his x2	2ml + 10µl	10ml + 100µl	*Start growing 9:30
his.SOD x2	2ml + 10µl	10ml + 100µl
yCCS x2	2ml + 10µl	10ml + 100µl

At OD=0.6 add IPTG /make glycerol stock (14:30)
- Sample 0h
  - 1ml culture
  - Spinn down 13000rpm, 5min, remove LB
  - Resuspend in 100µl sample buffer
  - Heat in 95°C, 5min
  - Freeze