"

From 2010.igem.org

Contents 1 Andreas 1.1 Cloning of N-CPPs into pSB1C3 & Extraction of RBS BioBrick (BBa_B0030) 1.1.1 Colony PCR 1.1.2 Gel verification 1.1.3 ON cultures 1.2 Extraction of RBS BioBrick (BBa_B0034) 1.3 Preparation of chemically competent Top10 2 Mimmi 2.1 Restriction enzymes control 2.1.1 Digestion 2.1.2 Gel 2.2 MITF-M 2.2.1 Site-Directed Mutagenesis

Andreas

Cloning of N-CPPs into pSB1C3 & Extraction of RBS BioBrick (BBa_B0030)

Transformations from 9/9 resulted in good colony yields on all plates. Chose "pSB1C3.N-CPP* 9/9" for colony PCR.

Colony PCR

Picked 12 N-CPP* clones (NC 1-12) and 4 RBS clones (RBS 1-4)

PCR tubes
dH2O	16.22
DreamTaq buffer	2
dNTPs, 10 mM	0.4
VF2	0.4
VR	0.4
DreamTaq pol.	0.08
Template DNA	0.5
	20 μl

PCR settings
Standard colony PCR protocol.

• 1:00 elongation

Gel verification

Also ran two samples for Mimmi (E & S)

1.5 % agarose, 90 V

Expected bands

• N-Tra10: 389 bp
• N-TAT: 359 bp
• N-LMWP: 368 bp
• RBS B0030: 253 bp

Results

• N-CPPs: Potentially correct bands for clones 2, 3, 5, 8, 9, 10, 11 & 12
• RBS B0030: Correct-sized bands for all four clones.

ON cultures

Set ON cultures for all relevant N-CPPs, for plasmid prep.

• N-CPP 2, 3, 5, 8, 9, 10, 11, 12
  • 5 ml LB + 25 Cm
  • 37 °C, 220 rpm

Selected clone 4 of RBS 30. Both plasmid prep and glycerol stock.

• RBS 30 4
  • 5 ml LB + 100 Amp
    • 37 °C, 220 rpm.
  • 3 ml LB + 100 Amp
    • 30 °C

Extraction of RBS BioBrick (BBa_B0034)

After studying the original RBS of our expression vector pEX, we decided that BBa_B0034 was a better candidate for our SOD/yCCS operon than BBa_B0030, as it better resembles the RBS of pEX, as well as minimizes the distance from the first gene in the operon.

Extracted BBa_B0034 (RBS 34), carried on pSB1A2, from iGEM plate 1, well 2M. Transformed into Top10.

• Quick transformation
• 1 μl DNA
• Amp 100

Preparation of chemically competent Top10

Since I've previously experienced slight AmpR contamination in our latest batch of competent Top10, I streaked an LB agar plate with competent Top10 cells to isolate single clones.

Plate grown ON in 37 °C.

Mimmi

Restriction enzymes control

Digestion

mix	(µl)		Conditions
sH2O	15		time	°C
10x buffer	3		30m	37
DNA (pSB1C3)	10		20m	65
enzyme (E/S)	1		OO	10	tot	29µl

Gel

well	sample
1	ladder
2	pSB1C3 cut E
3	pSB1C3 cut S

MITF-M

Site-Directed Mutagenesis

• Add Dpn1 (12.30-16=3.5h)
• Deactivate Dpn1 at 80°C for 20m