I am trying to make a 51 or 36 base pair stretch of semi-random mutagenesis. I have made a forward primer with the generic sequence (for the 51 bp mutation):
5’-X(8)-N(51)-Y(20)-3’
Where X(8) is 8 bases that are complementary to the 5’-end of where I want my mutations; N(51) is a semi-random sequence (85% same sequence as template, but ‘doped’ with 5 % of each of the other 3 bases in each position); Y(20) is a 20 bp sequence that anneals with the 3’-end of where I want my mutations (Tm ~60°C).
The reverse primer has a 5’-end complementary to the X(8) sequence on the forward, and another ~20 bp annealing to the template (no mutations in reverse primer).
This will make a PCR band, which is the entire plasmid (~7 kb), with 8 bp overhangs (kind of like USER cloning). Using PfuUltra II, the PCR gives a nice band of the right size, but I get nothing when transforming into coli.
I have tried using gel purified or PCR mix (both after DpnI treatment), I have tried TOP10 and DH5α cells. I am using amp resistance, and 100 µg/ml amp in my plates, and I do get loads of colonies with a template transformation.
I have attached a crude image of the PCR strategy, in case it’s easier than just reading it. Blue is the X(8) sequence, red is N(51) and green is Y(20). The first plasmid is the template and the primers, the second is the PCR product (which is linear, but with 8 bp overhang).
You could use phosphorylated primers and then self-ligate the PCR product. The plasmid length is 7kb and you've already demonstrated that you can perform PCR on the whole plasmid, so there is no reason to assume that the reaction wouldn't work with phosphorylated primers. The primers are a bit more expensive but you can skip the DpnI digest and the reaction is very reliable.
Hi Maximilian,
Thanks for the suggestion. I was actually kicking myself when they arrived because I forgot to order them phosphorylated. The random bases makes the price so high that they'd probably give me free phosphorylation. I did also consider phosphorylating them myself (as a backup plan), but I just can't see why in vivo ligation wouldn't work as well?
Did you try to transform your bacteria with the linear PCR product? You said " the PCR gives a nice band of the right size". If I understand your image correctly you should get a nicked circular plasmid which run at a different size than the linear product.
I only have a linear product (except from the template, which I digest with DpnI, so only 1 single distinct band on the gel); the pcr creates a plasmid corresponding to the lower one on the attached image. So linear with the 8 bp overhang. Or 'nicked' on both strands, with 8 bp between the 'nicks'.
Most likely the bacteria just don't 'like' your double 'nicked' plasmid. You could try your luck by just using a lot more DNA for transformation but even then I wouldn't expect more than a few colonies which doesn't help you if you want to check several mutants.
I just tried, from, yesterday to today, a transformation with several 100 ng PCR product, but still no colonies. When I have the time, I'll try phosphorylating the primers. I just don't see why it would not be possible? This (the linear DNA with 8 bp overhangs for in vivo ligation) is how USER cloning is usually done, right?
Update, for anyone stumbling into this post; Waiting for T4 PNK to arrive, I did my PCR (using Pfu Ultra II) again, but lowered my extension temperature from 72°C to 68°C, and increased my extension time from 1:50 to 4:30. I got 'dirtier' bands, but after gel purification and transformation, I have confirmed 14 clones with 1 - 8 mutations in each construct and 1 with no mutations (which is probably not template just one of the low % of primers that is identical to template). Now I'll just scale up.
Thanks again for the suggestions, Maximillian.
Cheers,
W
Please see the papers for your reference
https://www.researchgate.net/publication/311668087_Improvement_of_chitinase_Pachi_with_nematicidal_activities_by_random_mutagenesis
https://www.researchgate.net/publication/289367579_Characterization_and_directed_evolution_of_BliGO_a_novel_glycine_oxidase_from_Bacillus_licheniformis
https://www.researchgate.net/publication/282153514_Enhanced_nematicidal_potential_of_the_chitinase_pachi_from_Pseudomonas_aeruginosa_in_association_with_Cry21Aa
https://www.researchgate.net/publication/277326677_Improvement_of_glycine_oxidase_by_DNA_shuffling_and_site-saturation_mutagenesis_of_F247_residue
Regards
Article Improvement of chitinase Pachi with nematicidal activities b...
Article Characterization and directed evolution of BliGO, a novel gl...
Article Enhanced nematicidal potential of the chitinase pachi from P...
Article Improvement of glycine oxidase by DNA shuffling, and site-sa...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning