A while back I wrote a post on a T4 DNA polymerase dependent ligation independent cloning method. In the comments, Max asked if anyone had a protocol. Since there does not appear to be a simplified protocol available on the web, I thought I would post mine for reference.
It is adapted from a 2006 Organic & Biomolecular Chemistry article by Bonsor et al and although it works for me, I have to caution that I have not been using it for very long so improvements may be possible. If you have any suggestions on how the protocol can be changed or improved, let me know.
The protocol just deals with the bare bones of the digest, T4 DNA polymerase treatment, annealing and transformation. For information in designing the cloning site, see my original article, or the paper.
Here’s the protocol:
Vector Preparation
” Digest: 10 micrograms vector/10U enzyme, 20 microlitres reaction volume for 2hr.
” Separate on agarose gel and extract band
” T4 polymerase treatment
- 0.4 pmol digested vector
- 4 microlitres of reaction buffer
- 4 microlitres of 25mM dTTP
- 2 microlitres of 100mM DTT
- 20U T4 DNA polymerase
- Water to 40 microlitres
- Incubate 30min at 22°C
- Incubate 20min at 75°C (to stop reaction)
” Clean up reaction with PCR purification kit
Insert preparation
” After the PCR reaction, use a PCR purification kit to clean up.
” T4 polymerase treatment
- 0.2 pmol PCR product
- 2 microlitres T4 pol buffer
- 2 microlitres of 25mM dATP
- 1 microlitres 100mM DTT
- 10U T4 DNA pol
- Water to 20 microlitres
- Incubate at 30min at 22°C
- Incubate at 75°C 20min (to stop reaction)
” Clean up reaction with PCR purification kit
Anneal insert and vector
” Combine 2 microlitres insert + 1 microlitre vector
” Incubate at RT for 10min
” + 2 microlitres 100mM EDTA
” Heat to 75°C and cool slowly to RT
” Transform 2 microlitres as normal
