Koeris/Notebook/2007-1-19: Difference between revisions
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*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 for 2h | *Digest PCR amplicons with KpnI, Hind III - double digest for 2h | ||
**Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> | **Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> check cut sites. | ||
Used wrong enzyme - hipA has a cut site for Hind III | |||
Revision as of 08:37, 20 January 2007
DNA Fragment Purification from Acrylamide
Solutions
Crush and Soak Solution
- 500 mM NH4OAc 3.3 g NH4OAc
- 0.1% SDS 0.1 g SDS
- 0.1 mM EDTA 20 ml 500 mM EDTA
- up to 100 ml with Q
- store at room temperature
- 3 M NaOAc pH 5.2
- 24.6 g anhydrous sodium acetate
- pH to 5.2 with acetic acid and bring up to 100 ml with Q
- store at room temperature
Other Reagents
- DMCS treated glass wool
- 0.22 mm disposable micro tip filters (syringe type)
- blue tips with melted tips to serve as pestle for crushing acrylamide
Procedure
- Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band
- Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar.
- Add 1 ml crush and soak solution and incubate overnight at 37° C
- Spin in the microfuge for 10 minutes at 14,000 rpm
- Remove as much liquid as possible and KEEP IT
- Add another 500 microliters of crush and soak solution
- Repeat the spin and pool the recovered supernatant
- Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above)
- Spin as usual, wash and dry. Resuspend in 20 microliters TE (or Qiagen buffer EB)
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
- 92 deg C - 5min
- 92 deg C - 30s
- 50 deg C - 45s
- 72 deg C - 120s
- Cycle back to 2. 29X
- 72 deg C - 10min
- 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 for 2h
- Using Hind II might have been a mistake - a 200bp band showed up where it shouldn't have been -> check cut sites.
Used wrong enzyme - hipA has a cut site for Hind III
