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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of m-yCCS into pEX & Cloning of N-CPPs 1.1.1 Colony PCR 1.1.2 Gel verification 1.2 Cloning of N-CPPs into pSB1C3 1.2.1 Ligation 1.2.2 Transformation 1.2.3 Gel verification of digestion sample 1.3 Cloning of SOD⋅His into pMA 1.3.1 Sequencing results 2 Mimmi 2.1 Saftey issues on the home page 2.2 pMA.his.SOD 2.3 MITF-M 2.3.1 Colony PCR 2.4 pMA.his.SOD 2.4.1 Digestion 2.4.2 Gel

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

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 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.

1. 2 μl ligation mix†

   5 μl ligation mix

†: Possibly contaminated due to non-sterile transformation.

Gel verification of digestion sample

• 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.

Cloning of SOD⋅His into pMA

Sequencing results

• SH1: pMA.SOD⋅His 1 (fasta)
• SH2: pMA.SOD⋅His 2 (fasta)

Ran nucleotide Blasts (Blastn) to verify the sequencing results (SH1 Blastn / SH2 Blastn). Both Blasts revealed three identical silent mutations within the His tag. However, since they don't affect the amino acid sequence, and don't result in any rare codons, no action will be needed.

Mimmi

Saftey issues on the home page

• Making a first layout to answer the saftey questions...

pMA.his.SOD

• Plasmid prep. following E.Z.N.A. plasmid mini kit protocol 1
  • Wash two times with DNA wash buffer
  • Eluate in 50µl sH₂O

DNA concentration

266ng/µl

MITF-M

Colony PCR

Fermentas Pfu

Mix	(µl)	x5		Primers		conditions
sH2O	17	85		pSB_VF2		time	°C
10x buffer	2.5	12.5		pSB_VR		2m	95
dNTP	2.5	12.5				30s	95	)
F primer	1	5				30s	55	> 30 cycles
R primer	1	5				2m40s	72	)
DNA	0.5	5x0.5				10m	72
Pfu pol	0.5	2.5				oo	10
tot	25µl	125µl

pMA.his.SOD

Digestion

Mix	(µl)	(µl)		Conditions			[pMA.SOD.his] = 246ng/µl -> ~2µg = 8µl
DNA	8	7.5		Time	°C		[pMA.his.SOD] = 280ng/µl -> ~2µg = 7.5µl
s2O	17	17.5		30m	37		[pSB1C3] = 148ng/µl -> ~1.2µg = 8µl
10x buffer	3	3		20m	65
XbaI	1	1
PstI	1	1
tot	3x30µl

Gel

well	sample
1	1kb ladder
2	pMA.SOD.his
3	pMA.his.SOD
4	pSB1C3.RFP

• Cut gel bands

• Save in fridge