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From 2010.igem.org

Contents 1 Mimmi 1.1 MITF-M 1.1.1 Gel 2 Andreas 2.1 Cloning of N-CPPs into pSB1C3 2.1.1 Transformation results 2.1.2 Digestion 2.1.3 Control digestions 2.1.3.1 Gel verification 2.1.4 Ligation 2.1.5 Transformation 2.1.6 PCR

Mimmi

MITF-M

Gel

well	sample
1	1kb ladder
2	MITF-M 1
3	MITF-M 2
4	MITF-M 3
5	MITF-M 4
6	positive control
7	blank

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 3/9

More growth on the 2 μl plate than on the 5 μl plate, indicating what I already suspected: contamination in transformation. Discarded both plates and restarted cloning.

Digestion

[N-CPP plasmid] = 672 ng/μl

	N-CPP
10X buffer Tango	2
DNA (1 μg)	1.5
dH2O	12.5
XbaI	1
AgeI	4
	20 μl

Incubation: 37 °C, 3 h

Inactivation: 80 °C, 20 min

Control digestions

Did two control digestions to analyze the N-CPP plasmid vector size.

	A+S	ApaI
10X buffer Tango	2	2
DNA	1	1
dH2O	15	16
FD ApaI	1	1
FD SmaI	1	0
	20 μl	20 μl

Incubation: 37 °C, 30 min

Inactivation: 65 °C, 5 min

Gel verification

1 % agarose, 120 V, 40 min

• N-CPP: undigested N-CPP plasmid
• Dig X+A: N-CPP plasmid digested with XbaI & AgeI
• Dig ApaI: Linearized N-CPP plasmid digested with ApaI
• Dig S+A: N-CPP plasmid digested with SmaI and ApaI, excising the 391 bp N-CPP cluster.

Results
Linearized plasmid seems to be ≈5 kb long. Undigested plasmid moved slower; this was probably the result of the lane being too crowded.

Ligation

Vector: Dig pSB1C3 X+A EXTR 1

	1	2*
5X Rapid Ligation buffer	4	4
Vector DNA	4	4
Insert DNA	11	9
dH2O	0	2
T4 DNA ligase	1	1
	22 μl †	20 μl

† Accidentally added 2 μl FastAP alkaline phosphatase to ligation mix 1. Enzyme inactivated at 75 °C, 10 min, prior to ligation.

• Incubation: 22 °C, 10 min

Transformation

Performed by Mimmi

Standard transformation, except:

• 30 min incubation in 37 °C
• 3 μl ligation mix

Cells grown on Cm 25 plates.

PCR

Since we've experienced some problems with our N-CPP clonings, a PCR of each individual N-CPP was performed for individual cloning, in case the current cloning doesn't work.

Reactions & primers
	Forward	Reverse
N-Tra10	VF2	VR
N-TAT	pEXf	pEXr
N-LMWP	pGexf	pGexr
N-CPP cluster	VF2	pGexr

PCR tubes
10X Pfu buffer	5
dNTP, 10 mM	1
dH2O	41
Template DNA	0.5
Forw. primer	1
Rev. primer	1
Pfu DNA pol.	0.5
	50 μl

Standard colony PCR settings

• Elongation: 1:15

Tubes stored in -20 °C.