Team:Stockholm/9 June 2010

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I transformed both my own cells from yesterday and also the kit's competent cells. Each one had a two serial 10-fold dilution.
I transformed both my own cells from yesterday and also the kit's competent cells. Each one had a two serial 10-fold dilution.
 +
 +
----
 +
===Preparation LB agar plates with ampicillin resistance===
 +
 +
LB agar:
 +
*25 g LB
 +
*15 g bactoagar
 +
*Fill up to 1 L with distilled water
 +
 +
# Autoclave for 120°C, 20 min
 +
# Cool down
 +
# Add 200 mg ampiciliin in 1,8 ml water
 +
# Pour out on plates
 +
 +
= Johan =
 +
 +
== Transformation ==
 +
 +
A transformation was performed to test the competence of the cells, both with our own and competent cells from a kit.
 +
 +
== LB agar plates with ampicillin ==
 +
 +
* 25 g LB
 +
* 15 g bactoAgar
 +
* up to 1L ddH2O
 +
 +
* Autoclave 120 °C 20 min
 +
* Cool down
 +
* Add 200 mg Amp in 1,8 ml H2O
 +
* For 1L of LB add 1 ml of 100 mg/ml
 +
 +
* Pour on plates
 +
 +
== PCR ==
 +
 +
PCR of SOD and yCCS to add prefix/suffix
 +
 +
=== Mix ===
 +
 +
* 30 µl H2O
 +
* 10 µl 5x buffer
 +
* 1 µl 50 mM MgCl2
 +
* 1 µl 10 mM dNTPS
 +
* 1 µl DNA templ.
 +
* 3 µl 5 µM F.primer
 +
* 3 µl 5 µM R.primer
 +
* 1 µl PfuX Polymerase
 +
 +
=== Program ===
 +
 +
1: 98 °C 2 min
 +
 +
2: 98 °C 10 sec
 +
 +
3: 55 °C 15 sec
 +
 +
4: 72 °C 45 sec
 +
 +
2-4 repeat 30 times
 +
 +
5: 72 °C 5 min
 +
 +
6: 4 °C ∞
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:26, 26 October 2010

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Contents

Nina

Testing DH5α competent cells by transformation

This was performed according to tranformation protocol From NEB 5-alpha competent E.coli (High Efficiency) NEW ENGLAND BioLabs.

I transformed both my own cells from yesterday and also the kit's competent cells. Each one had a two serial 10-fold dilution.

Preparation LB agar plates with ampicillin resistance

LB agar:

  • 25 g LB
  • 15 g bactoagar
  • Fill up to 1 L with distilled water
  1. Autoclave for 120°C, 20 min
  2. Cool down
  3. Add 200 mg ampiciliin in 1,8 ml water
  4. Pour out on plates

Johan

Transformation

A transformation was performed to test the competence of the cells, both with our own and competent cells from a kit.

LB agar plates with ampicillin

  • 25 g LB
  • 15 g bactoAgar
  • up to 1L ddH2O
  • Autoclave 120 °C 20 min
  • Cool down
  • Add 200 mg Amp in 1,8 ml H2O
  • For 1L of LB add 1 ml of 100 mg/ml
  • Pour on plates

PCR

PCR of SOD and yCCS to add prefix/suffix

Mix

  • 30 µl H2O
  • 10 µl 5x buffer
  • 1 µl 50 mM MgCl2
  • 1 µl 10 mM dNTPS
  • 1 µl DNA templ.
  • 3 µl 5 µM F.primer
  • 3 µl 5 µM R.primer
  • 1 µl PfuX Polymerase

Program

1: 98 °C 2 min

2: 98 °C 10 sec

3: 55 °C 15 sec

4: 72 °C 45 sec

2-4 repeat 30 times

5: 72 °C 5 min

6: 4 °C ∞





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

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