Koeris/Notebook/2007-1-19

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(Gel image)
(Gel image)
Line 29: Line 29:
#4 deg C - indefinite
#4 deg C - indefinite
==Gel image==
==Gel image==
-
Loaded 5ul, i.e. 5% of Rx on the gel
+
Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well
==Restriction digest==
==Restriction digest==
*Use Qiagen PCR cleanup kit to remove salts and enzymes
*Use Qiagen PCR cleanup kit to remove salts and enzymes
*Digest PCR amplicons with KpnI, Hind III - double digest
*Digest PCR amplicons with KpnI, Hind III - double digest

Revision as of 14:00, 19 January 2007

Contents

Re-cloning of [His]6

Due to lack of material, I could not recover the A plasmid and have to re-clone it.

PCR set-up

  • 100 ul total reaction volume
  • 1ul template DNA @ ~40ng/ul
  • 200pmol primer mix
  • 97ul Taq SuperMix

Pipetting scheme

Labels in the respective field codes

Primer Tube Label
A his6 2F&R 200pmol A1
A his6 2F&R 200pmol A2

Thermal profile

  1. 92 deg C - 5min
  2. 92 deg C - 30s
  3. 50 deg C - 45s
  4. 72 deg C - 120s
  5. Cycle back to 2. 29X
  6. 72 deg C - 10min
  7. 4 deg C - indefinite

Gel image

Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well

Restriction digest

  • Use Qiagen PCR cleanup kit to remove salts and enzymes
  • Digest PCR amplicons with KpnI, Hind III - double digest
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