Team:Stockholm/17 August 2010

From 2010.igem.org

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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.
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.
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== Mimmi ==
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=== MITF ===
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==== pRc/CMV plasmid control ====
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{|
 +
! Mix
 +
| (µl)
 +
| X8
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| rowspan="9" |
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! primers
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|-
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| sH<sub>2</sub>O
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| 18.75
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| 150
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| MITF_F
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|-
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| F primer
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| 1.25
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| 10
 +
| MITF_R
 +
|-
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| R primer
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| 1.25
 +
| 10
 +
| rowspan="6" |
 +
|-
 +
| dNTP
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| 0.5
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| 4
 +
|-
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| 10X buffer
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| 5
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| 20
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|-
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| Pfu turbo
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| 0.25
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| 2
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|-
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| DNA
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|0.5
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| 8X0.5
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|-
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| align="right" | tot
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| 25µl
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| 200µl
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|}
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 +
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{|
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! colspan="2" | conditions
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| rowspan="3" width="150" |
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! colspan="2" | conditions
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| rowspan="3" width="150" |
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! colspan="2" | conditions
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| rowspan="3" width="150" |
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|-
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! time
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! &deg;C
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! time
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! &deg;C
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! time
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! &deg;C
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|-
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| 2m
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| 94
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| 2m
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| 94
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| 2m
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| 94
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|-
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| 30s
 +
| 94
 +
| )
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| 30s
 +
| 94
 +
| )
 +
| 30s
 +
| 94
 +
| )
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|-
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| 30s
 +
| 45, 49.3, 55 
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| > 5 cycles
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| 1m
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| 50
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| > 5 cycles
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| 1m
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| 50
 +
| > 30 cycles
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|-
 +
| 1m30s
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| 72
 +
| )
 +
| 1m30s
 +
| 72
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| )
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| 1m30s
 +
| 72
 +
| )
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|-
 +
| 30s
 +
| 94
 +
| )
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| 30s
 +
| 94
 +
| )
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| 10m
 +
| 72
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| rowspan="5" |
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|-
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| 30s
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| 65
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| > 25 cycles
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| 1m
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| 65
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| > 25 cycles
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| oo
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| 10
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|-
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| 1m30s
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| 72
 +
| )
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| 1m30s
 +
| 72
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| )
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| colspan="2" rowspan="3" |
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|-
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| 10m
 +
| 72
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| rowspan="2" |
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| 10m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 10
 +
| oo
 +
| 10
 +
|}

Revision as of 08:21, 20 August 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

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Contents

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:

Bild5.jpg

Bild9.jpg

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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