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Team:Stockholm/17 August 2010
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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 | |||||
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