Team:Stockholm/22 July 2010

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m (→Hassan)
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The ladder is FastRuler DNA ladder low range, but the bands does not show the expected size. Did I mistakenly use the FastRuler DNA ladder middle range?
The ladder is FastRuler DNA ladder low range, but the bands does not show the expected size. Did I mistakenly use the FastRuler DNA ladder middle range?
 +
 +
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 +
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== Mimmi ==
 +
 +
=== LMWP ===
 +
 +
==== glycerol stock + preparation of plasmids ====
 +
 +
 +
{|
 +
! Mix
 +
|
 +
|-
 +
| LB
 +
| 12ml
 +
|-
 +
| Amp
 +
| 24µl
 +
|-
 +
| ON culture
 +
| 120µl
 +
|}
 +
 +
 +
 +
*Shake in 37°C, ~200rpm, ~3h
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 +
*Add 1600µl culture to pre-made jar with 400µl sterile glycerol
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 +
*Take 5ml culture and follow the mini plasmid prep. kit 1 protocol.
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 +
**Wash 2X with EtOH solution
 +
 +
**Dilute in 50µl HPL6 H<sub>2</sub>O
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 +
:--> in the freezer (plasmid & DNA ) pEX.BBa_J18930A -> 32A
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 +
 +
 +
 +
==== ligation ====
 +
 +
 +
{|
 +
| LMWP_N_F1
 +
| 2µg/µl
 +
|-
 +
| LMWP_CN_F2
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| 1.6µg/µl
 +
|-
 +
| LMWP_CN_R1
 +
| 2µg/µl
 +
|-
 +
| LMWP_N_R2
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| 1.5µg/µl
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|-
 +
|
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|
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|-
 +
| LMWP_C_F1
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| 1.8µg/µl
 +
|-
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| LMWP_C_R2
 +
| 1.5µg/µl
 +
|}
 +
 +
 +
 +
 +
 +
{|
 +
! Mix
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| (µl)
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|-
 +
| F1
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| 1
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|-
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| F2
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| 1
 +
|-
 +
| R1
 +
| 1
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|-
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| R2
 +
| 1
 +
|-
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| H<sub>2</sub>O
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| 4
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|-
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| DNA sol.
 +
| 42
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|-
 +
| align="right" | tot
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| 50µl
 +
|}
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*Heatshock 90&deg;C 3min
 +
 +
*Incubate 37&deg;C 1h
 +
 +
 +
 +
{|
 +
! Mix
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|
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| 3X
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|-
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| H<sub>2</sub>O
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| 5
 +
| rowspan="6" |
 +
|-
 +
| plasmid
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| 2
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|-
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| T4 buffer
 +
| 1
 +
|-
 +
| T4 ligase
 +
| 1
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|-
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| DNA
 +
| 1
 +
|-
 +
| align="right" | tot
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| 10µl
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|}
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 +
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*Incubate 22&deg;C 1-3h
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*Thaw 3x100µl Top10 competent cells
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*Add 1µl plasmid
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*Hold on ice 30min
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*Heat shock 42&deg;C 50s
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*Cool down on ice
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*Add 900µl LB
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:Incubate 37&deg;C 1h 250rpm
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*Spin down cells 13000rpm 15s
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*Remove 900µl, resuspend in remaining 100µl
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*Plate 100µl on LB agar plates with Cm<sup>r</sup>
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*Grow in 37&deg;C ON

Revision as of 20:16, 29 August 2010

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↳   Notebook Safety Protocols


Contents


Hassan

  • tyrp1
  • nalp1
  • mitf
  • CGRP
  • S100B
  • foxd3
  • GGA1
  • LMP7
  • TAPBP

Johan

PCR

The PCR samples that was run on a gel to verify that the colonies had been successfully site-directed mutagenesised seemed to be lost, and a new colony PCR was performed to verify the site-directed mutagenesis before cleaving and adding it to the target vectors.

Agarose Johan bFGF 22july 22july.jpg

The ladder is FastRuler DNA ladder low range, but the bands does not show the expected size. Did I mistakenly use the FastRuler DNA ladder middle range?



Mimmi

LMWP

glycerol stock + preparation of plasmids

Mix
LB 12ml
Amp 24µl
ON culture 120µl


  • Shake in 37°C, ~200rpm, ~3h
  • Add 1600µl culture to pre-made jar with 400µl sterile glycerol
  • Take 5ml culture and follow the mini plasmid prep. kit 1 protocol.
    • Wash 2X with EtOH solution
    • Dilute in 50µl HPL6 H2O
--> in the freezer (plasmid & DNA ) pEX.BBa_J18930A -> 32A



ligation

LMWP_N_F1 2µg/µl
LMWP_CN_F2 1.6µg/µl
LMWP_CN_R1 2µg/µl
LMWP_N_R2 1.5µg/µl
LMWP_C_F1 1.8µg/µl
LMWP_C_R2 1.5µg/µl



Mix (µl)
F1 1
F2 1
R1 1
R2 1
H2O 4
DNA sol. 42
tot 50µl


  • Heatshock 90°C 3min
  • Incubate 37°C 1h


Mix 3X
H2O 5
plasmid 2
T4 buffer 1
T4 ligase 1
DNA 1
tot 10µl


  • Incubate 22°C 1-3h
  • Thaw 3x100µl Top10 competent cells
  • Add 1µl plasmid
  • Hold on ice 30min
  • Heat shock 42°C 50s
  • Cool down on ice
  • Add 900µl LB
Incubate 37°C 1h 250rpm
  • Spin down cells 13000rpm 15s
  • Remove 900µl, resuspend in remaining 100µl
  • Plate 100µl on LB agar plates with Cmr
  • Grow in 37°C ON
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