Team:Stockholm/17 August 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==)
 
(3 intermediate revisions not shown)
Line 2: Line 2:
==Andreas==
==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====
 +
{|border="1" cellpadding="2" cellspacing="0" align="right"
 +
!colspan="5"|DNA concentrations
 +
|-
 +
! 
 +
!colspan="2"|Prior to concentration
 +
!colspan="2"|After concentration
 +
|-
 +
!Sample
 +
!Conc [ng/μl]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
!Conc [ng/&mu;l]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|yC1&rarr;pSB1C3.m-yCCS 1
 +
|36.88
 +
|1.82
 +
|250.2
 +
|1.92
 +
|-
 +
|yC4&rarr;pSB1C3.m-yCCS 4
 +
|41.83
 +
|1.77
 +
|215.7
 +
|1.87
 +
|-
 +
|SC1&rarr;pSB1C3.m-SOD 1
 +
|47.38
 +
|1.72
 +
|230.2
 +
|1.91
 +
|-
 +
|SC2&rarr;pSB1C3.m-SOD 2
 +
|38.00
 +
|1.72
 +
|104.0
 +
|1.86
 +
|}
 +
''From 16/8 ON cultures''
 +
 +
Elution volume: 70 &mu;l dH<sub>2</sub>O
 +
 +
Since samples were intended for sequencing, they had to be concentrated in the evaporator. Results in table.<br />
 +
Samples sent for sequencing from VF2 primer annealing site:
 +
*15 &mu;l DNA
 +
*1.5 &mu;l 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:
 +
 +
[[Image:Bild5.jpg]]
 +
 +
[[Image:Bild9.jpg|350px]]
 +
 +
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
 +
| rowspan="9" |
 +
! primers
 +
|-
 +
| sH<sub>2</sub>O
 +
| 18.75
 +
| 150
 +
| MITF_F
 +
|-
 +
| F primer
 +
| 1.25
 +
| 10
 +
| MITF_R
 +
|-
 +
| R primer
 +
| 1.25
 +
| 10
 +
| rowspan="6" |
 +
|-
 +
| dNTP
 +
| 0.5
 +
| 4
 +
|-
 +
| 10X buffer
 +
| 5
 +
| 20
 +
|-
 +
| Pfu turbo
 +
| 0.25
 +
| 2
 +
|-
 +
| DNA
 +
|0.5
 +
| 8X0.5
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| 200µl
 +
|}
 +
 +
 +
 +
{|
 +
! colspan="2" | conditions
 +
| rowspan="3" width="150" |
 +
! colspan="2" | conditions
 +
| rowspan="3" width="150" |
 +
! colspan="2" | conditions
 +
| rowspan="3" width="150" |
 +
|-
 +
! time
 +
! &deg;C
 +
! time
 +
! &deg;C
 +
! time
 +
! &deg;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
 +
| rowspan="5" |
 +
|-
 +
| 30s
 +
| 65
 +
| > 25 cycles
 +
| 1m
 +
| 65
 +
| > 25 cycles
 +
| oo
 +
| 10
 +
|-
 +
| 1m30s
 +
| 72
 +
| )
 +
| 1m30s
 +
| 72
 +
| )
 +
| colspan="2" rowspan="3" |
 +
|-
 +
| 10m
 +
| 72
 +
| rowspan="2" |
 +
| 10m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 10
 +
| oo
 +
| 10
 +
|}
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:56, 26 October 2010

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

↳   Notebook Safety Protocols


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





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/17_August_2010"