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Latest revision as of 10:56, 26 October 2010

Contents 1 Andreas 1.1 Site-directed mutagenesis 1.1.1 Glycerol stocks 1.1.2 Plasmid prep 1.1.3 ON cultures 2 Nina 2.1 Colony PCR 3 Mimmi 3.1 MITF 3.1.1 pRc/CMV plasmid control

Andreas

Site-directed mutagenesis

Continued from 16/8

Glycerol stocks

From 16/8 ON cultures

yC1: pSB1C3.m.yCCS 1, 17/8 yC4: pSB1C3.m.yCCS 4, 17/8 SC1: pSB1C3.mut SOD 1, 17/8 SC2: pSB1C3.mut SOD 2, 17/8

1600 μl cell culture in 400 μl 100 % glycerol

Plasmid prep

DNA concentrations
	Prior to concentration		After concentration
Sample	Conc [ng/μl]	A260/A280	Conc [ng/μl]	A260/A280
yC1→pSB1C3.m-yCCS 1	36.88	1.82	250.2	1.92
yC4→pSB1C3.m-yCCS 4	41.83	1.77	215.7	1.87
SC1→pSB1C3.m-SOD 1	47.38	1.72	230.2	1.91
SC2→pSB1C3.m-SOD 2	38.00	1.72	104.0	1.86

From 16/8 ON cultures

Elution volume: 70 μl dH₂O

Since samples were intended for sequencing, they had to be concentrated in the evaporator. Results in table.
Samples sent for sequencing from VF2 primer annealing site:

• 15 μl DNA
• 1.5 μl 10 mM VF2 primer

ON cultures

New ON cultures (5 ml + 25 Cm) were set for plasmid prep from same clones as were sent for sequencing: yC1, yC4, SC1, SC2.

Nina

Colony PCR

I ran the colony PCR from yesterday on an agarose gel 1 % 100 V to screen whether or not there are any successful fusion proteins made up by protein A and IgG protease.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arragement on the gel:

Unfortunately I did not obtain any good bands on the gel that would represent the correct gene size. This must mean that something might have gone wrong with the ligation and therefore I will redo the ligation. In addition I will perform a gel clean up of the digested samples in order to not have any material interfering with the ligation.

Mimmi

MITF

pRc/CMV plasmid control

Mix	(µl)	X8		primers
sH2O	18.75	150		MITF_F
F primer	1.25	10		MITF_R
R primer	1.25	10
dNTP	0.5	4
10X buffer	5	20
Pfu turbo	0.25	2
DNA	0.5	8X0.5
tot	25µl	200µl

conditions			conditions			conditions
time	°C		time	°C		time	°C
2m	94		2m	94		2m	94
30s	94	)	30s	94	)	30s	94	)
30s	45, 49.3, 55	> 5 cycles	1m	50	> 5 cycles	1m	50	> 30 cycles
1m30s	72	)	1m30s	72	)	1m30s	72	)
30s	94	)	30s	94	)	10m	72
30s	65	> 25 cycles	1m	65	> 25 cycles	oo	10
1m30s	72	)	1m30s	72	)
10m	72		10m	72
oo	10		oo	10