I am trying to stitch in a 38 amino acid tag to the N-terminal end of my protein (3200bp) to be cloned into a lentiviral vector (~7000bp). The forward primer for the same, along with the overhang and the restriction site, comes about 150bp long. The first round of amplication gives me a band close to about 3000-3500bp along with a lot of other non specific bands at the higher molecular weight range. I then gel elute this specific band and reamplify using it as a template with the same primers but i end up getting a smear on the gel. I have also tried using this gel eluted sample to proceed with the digestion and ligation with my vector but in vain.
My PCR parameters are as follows:
1. 98 degC- 2min
2. 98 degC- 10s
3. 65 degC- 30s (2-4: x25 cycles)
4. 72 degC- 2min
5. 72 degC- 5min
6. 4 degC- hold
I use Q5 polymerase (strangely, I do not get any amplification with Phusion). I have tried a gradient PCR and it generally works in the range of (58-68 degC). I use about 50ng of the plasmid template for amplification. I understand that really long primers hamper the quality of amplification but unfortunately, this is a necessity right now.
I would really appreciate if anyone with experience can help me out here. My molecular biology is not THAT strong so please point out if I am committing any obvious mistakes.
Thanks in advance!
I am interested in your statement that you re amplified with the same primers. There is a risk of over amplification here.If every 10 cycles makes 1000 time more product then the product rapidly overwhelms the primer and you can get smears of concatomers where the product self anneals and extends with other product. For re amplification I would try 6 tubes . dilute the first product 1in 100 and cycle removing 1 tube every 2 cycles between 10 and about 18 cycles and see if any of these gives a clean product. With primers that long self annealing hairpins may be a problem so you could try the pcr with 1M betaine final concentration or up to 5% DMSO to avoid self annealing although DMSO at very high temperatures can denature the enzyme
Dear Dr. Rutland
Thank you for your answer. It makes sense that overamplication is what caused them to turn smeary. I will try dilutions as you recommended. Also, I have tried 3% DMSO in all my PCRs. Will try betaine as well.
Regards
Radhika
I have experience in PCR with long primers (not 150 but 110), using PfuII polymerase. I'm wondering why do you have to do a second round of PCR. In my case, when I needed to amplify a similar insert (3500 pb) with one round of PCR was enough for the cloning.
I think you might be able to do just one PCR and changing cycles from 25 to 35 to obtain more insert ratio (I've checked that with your polymerase it is possible to do so). Another thing that might improve the outcome is not to do the gel elution but direct elution from the PCR mix. If you are using only 50ng of the template-backbone you should not have problems if you purify all the PCR and use that purified insert directly for the next step (I only do gel extraction for the backbone after the digestion, but never for the inserts, and I have never had problems).
Gel-extraction kits normally allow direct PCR clean-up, and that increases the recovery efficiency quite a lot, affording you the second round of PCR for the insert.
I hope it helps! Good luck!
Ale
Dear Dr. Pazo
Thank you for your answer. Initially, I processed my pcr products after only one round of amplification. But I did purify (gel elute) at every step since there was a long of non specific bands as well. The idea of a second round was to try to reduce this non-specificity. However, I can try your suggestion of using the PCR product directly for disgestion.
Cheers
Radhika
Another strategy would be to order a forward primer and then the reverse primer of your insert of interest. The two primers would constitute your insert of interest. Fuse the two primers and phosphorylate the insert (the primers can also be phosphorylated before the fusion).
You then open your lentiviral vector with a restriction enzyme (preferably one that create a blunt-end) and if that is not the case use another site and blunt it using Klenow (dephosphorylate the vector before ligation).
Ligate the insert and vector.
That should do the trick. Now you just need to ensure that you only have one insert and it is in frame. Alternatively, you can also include new restriction sites in your primers so it will match your restriction sites in the vector.
Happy cloning and good luck, Simon
In my case, when I have to clone a tag, instead of using a long primer I prefer using a first couple of primers to amplified the region of interest and a first part of the tag, and then directly a second PCR whit a 2nd forward primer to match the first part of the tag and then add a second part of the tag and so on. In this way you can split the long primer.
This strategy worked well to have 20 aa tag, maybe it would work also for you!
Good luck
Martina
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