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From 2010.igem.org

Contents 1 Andreas 1.1 Assembly of new parts 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.3.1 Gel 1 1.1.3.2 Gel 2 1.1.3.3 Gel 3 1.2 Plasmid prep 1.3 Assembly of His⋅SOD⋅C-TAT 1.3.1 Digestion 1.3.2 Ligation 1.3.3 Transformation 1.4 ON cultures 2 Mimmi 2.1 SOD.his / his.SOD / yCCS 2.1.1 glycerol stock / over expression

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

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

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

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

• Sample 1h

• Sample 2h

• Sample 3h