Team:Stockholm/3 September 2010

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Andreas

Transfer of m-yCCS into pEX & Cloning of N-CPPs

Restreak results from 2/9 revealed four white (positive) clones. These were picked for colony PCR (y5, y6, y8 and y12).
Also picked four colonies from N-CPP plate from 30/8 (NC1, NC2, NC3 and NC4).

Colony PCR

PCR tubes
dH₂O	16.2
DreamTaq buffer	2
10 mM dNTPs	0.4
pEXf	0.4
pEXr	0.4
DNA	0.5
DreamTaq pol.	0.08
Total	20 μl

Gel verification

Gel verification of pSB1C3.N-CPPs and pEX.yCCS colony PCR.
3 μl λ; 5 μl sample.
λ=O'GeneRuler 1 kb DNA ladder

1 % agarose, 80 V

Expected bands

pSB1C3.N-CPPs: 365 bp (TAT), 374 bp (LMWP), 395 bp (Tra10)
pEX.m-yCCS: 963 bp

Results
Correct bands for pEX.m-yCCS clones 5, 6 and 8. No band for y12. 5 & 8 will later be selected for plasmid prep.
Too large bands for all CPP clones to be correct, which is strange. New cloning will be attempted.

Cloning of His⋅SOD into pMA

Plasmid prep

From 2/9 ON cultures
Elution: 50 μl x2

DNA concentrations
Sample	Conc. [ng/μl]	A₂₆₀/A₂₈₀
pSB1C3.m-yCCS 1	143.4	1.91

Sequencing

pSB1C3.m-yCCS: ASB0045 A55

Glycerol stock

pMA.His⋅SOD 2010-09-03

Cloning of N-CPPs into pSB1C3

Re-ligation of 30/8 digestion.

Ligation

[Dig. pSB1C3 X+A EXTR 1] = 13.72 ng/μl [Dig. N-CPP X+A] = 66.6 ng/μl

Ligation mix
Vector DNA	6
Insert DNA	9
5X Rapid Ligation buf.	4
dH₂O	0
T4 DNA ligase	1
Total	20 μl

Transformation

Standard transformation procedures.

2 μl ligation mix†
5 μl ligation mix

†: Possibly contaminated due to non-sterile transformation.

Gel verification of digestion sample

Gel verification of undigested and digested (XbaI & AgeI) N-CPP cluster plasmid.
3 μl λ; 3 μl sample
1 kb λ=O'GeneRuler 1 kb DNA ladder; 50 bp λ=GeneRuler 50 bp DNA ladder

NCPP: undigested N-CPP cluster plasmid
Dig NCPP: digested N-CPP cluster plasmid, XbaI and AgeI

1 % agarose, 100 V

Results
Difficult to interpret results, since the source plasmid size is unknown. However, it looks like the digested sample presents a shorter fragment compared to the undigested. On the other hand, it also looks like the size difference is too big to have resulted from just excising the ≈300 bp N-CPP cluster.
Also strange is that it looks like there are two bands present in the undigested sample.