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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 His⋅SOD into pMA 1.2.1 Plasmid prep 1.2.2 Sequencing 1.2.3 Glycerol stock 1.3 Cloning of N-CPPs into pSB1C3 1.3.1 Ligation 1.3.2 Transformation 1.3.3 Gel verification of digestion sample 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 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.

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.

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