Team:Stockholm/13 September 2010

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==Andreas==
==Andreas==
 +
===Preparation of Top10 chemically competent cells===
 +
None of the clones streaked onto the Amp 100 plate (11/9) grew, indicating no AmpR contamination in cells. One of the ON cultures were therefore selected for preparation of competent cells.
 +
 +
Procedures according to protocol. Growth conditions changed to 25 &deg;C, 220 rpm (OD<sub>600</sub> reached after &asymp;6 h).
 +
 +
A 100 &mu;l aliquot was divided and spread onto Amp 100, Cm 25 and Km 50 plates to verify that they were not contaminated.
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
Sequencing results from 8/9 returned.
 +
*pSB1C3.nCPP 3 (failed)
 +
*pSB1C3.nCPP 4 ([[media:PSB1C3.nCPP_4_premix.txt|fasta]])
 +
*pSB1C3.nCPP 6 ([[media:PSB1C3.nCPP*_6_premix.txt|fasta]])
 +
*pSB1C3.nCPP 7 ([[media:PSB1C3.nCPP*_7_premix.txt|fasta]])
 +
*pSB1C3.nCPP 8 ([[media:PSB1C3.nCPP*_8_premix.txt|fasta]])
 +
 +
Blastn alignments against [[media:Blastn_N-Tra10_pSB1C3.nCPP_11sep.txt|N-Tra10]], [[media:Blastn_N-TAT_pSB1C3.nCPP_11sep.txt|N-TAT]] and [[media:Blastn_N-LMWP_pSB1C3.nCPP_11sep.txt|N-LMWP]] indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).
 +
 +
====Transformations====
 +
Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.
 +
 +
Standard transformation with 1 &mu;l plasmid DNA.
 +
*pSB1C3.N-TAT
 +
*pSB1C3.N-Tra10
 +
 +
====Sequencing====
 +
DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.
 +
*'''pSB1C3.nCCP 2''': ABS0045 B92
 +
*'''pSB1C3.nCCP 3''': ABS0045 B93
 +
*'''pSB1C3.nCCP 5''': ABS0045 B94
 +
*'''pSB1C3.nCCP 8''': ABS0045 B95
 +
*'''pSB1C3.nCCP 9''': ABS0045 B96
 +
*'''pSB1C3.nCCP 10''': ABS0045 B97
 +
*'''pSB1C3.nCCP 11''': ABS0045 B98
 +
*'''pSB1C3.nCCP 12''': ABS0045 B99
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== MITF-M ===
 +
 +
==== Site-Directed Mutagenesis control ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| rowspan="7" width="100" |
 +
! colspan="2" | Conditions
 +
|-
 +
| DNA
 +
| 20
 +
! time
 +
! &deg;C
 +
|-
 +
| 10x buffer
 +
| 3
 +
| 3h
 +
| 37
 +
|-
 +
| sH<sub>2</sub>O
 +
| 5
 +
| colspan="2" rowspan="4" |
 +
|-
 +
| XbaI
 +
| 1
 +
|-
 +
| AgeI
 +
| 1
 +
|-
 +
| align="right" | tot
 +
| 30µl
 +
|}
 +
 +
 +
 +
==== Gel ====
 +
 +
[[Image:2010-09-13_MITF_SD1.jpg|200px|thumb|left|]]
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{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| MITF-col 1
 +
|-
 +
| 3
 +
| MITF-col 1 cut X+A
 +
|-
 +
| 4
 +
| MITF-col 2
 +
|-
 +
| 5
 +
| MITF-col 2 cut X+A
 +
|-
 +
| 6
 +
| MITF-col 3
 +
|-
 +
| 7
 +
| MITF-col 3 cut X+A
 +
|-
 +
| 8
 +
| MITF-col 4
 +
|-
 +
| 9
 +
| MITF-col 4 cut X+A
 +
|-
 +
| 10
 +
| MITF-M
 +
|-
 +
| 11
 +
| MITF-M cut X+A
 +
|}
 +
 +
*Should have saved more original MITF (stupid!)
 +
**Make new MITF-M
 +
 +
=== RBS ===
 +
 +
==== plasmid prep. ====
 +
 +
*Follow original protocol
 +
**Wash 2 times with DNA wash buffer
 +
**Eluate in 50µl
 +
**Eluate 2 times with the same sH<sub>2</sub>O
 +
 +
'''DNA conc.'''
 +
RBS34a ~42ng/µl -> ~60ng/µl
 +
RBS34b ~41ng/µl -> ~60ng/µl
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:03, 26 October 2010

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Contents

Andreas

Preparation of Top10 chemically competent cells

None of the clones streaked onto the Amp 100 plate (11/9) grew, indicating no AmpR contamination in cells. One of the ON cultures were therefore selected for preparation of competent cells.

Procedures according to protocol. Growth conditions changed to 25 °C, 220 rpm (OD600 reached after ≈6 h).

A 100 μl aliquot was divided and spread onto Amp 100, Cm 25 and Km 50 plates to verify that they were not contaminated.

Cloning of N-CPPs into pSB1C3

Sequencing results from 8/9 returned.

  • pSB1C3.nCPP 3 (failed)
  • pSB1C3.nCPP 4 (fasta)
  • pSB1C3.nCPP 6 (fasta)
  • pSB1C3.nCPP 7 (fasta)
  • pSB1C3.nCPP 8 (fasta)

Blastn alignments against N-Tra10, N-TAT and N-LMWP indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).

Transformations

Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.

Standard transformation with 1 μl plasmid DNA.

  • pSB1C3.N-TAT
  • pSB1C3.N-Tra10

Sequencing

DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.

  • pSB1C3.nCCP 2: ABS0045 B92
  • pSB1C3.nCCP 3: ABS0045 B93
  • pSB1C3.nCCP 5: ABS0045 B94
  • pSB1C3.nCCP 8: ABS0045 B95
  • pSB1C3.nCCP 9: ABS0045 B96
  • pSB1C3.nCCP 10: ABS0045 B97
  • pSB1C3.nCCP 11: ABS0045 B98
  • pSB1C3.nCCP 12: ABS0045 B99



Mimmi

MITF-M

Site-Directed Mutagenesis control

mix (µl) Conditions
DNA 20 time °C
10x buffer 3 3h 37
sH2O 5
XbaI 1
AgeI 1
tot 30µl


Gel

2010-09-13 MITF SD1.jpg
well sample
1 ladder
2 MITF-col 1
3 MITF-col 1 cut X+A
4 MITF-col 2
5 MITF-col 2 cut X+A
6 MITF-col 3
7 MITF-col 3 cut X+A
8 MITF-col 4
9 MITF-col 4 cut X+A
10 MITF-M
11 MITF-M cut X+A
  • Should have saved more original MITF (stupid!)
    • Make new MITF-M

RBS

plasmid prep.

  • Follow original protocol
    • Wash 2 times with DNA wash buffer
    • Eluate in 50µl
    • Eluate 2 times with the same sH2O

DNA conc. RBS34a ~42ng/µl -> ~60ng/µl RBS34b ~41ng/µl -> ~60ng/µl





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

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