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#1 Re: rf-cloning troubles » Problem with Secondary PCR » 2017-03-10 06:12:40
Dear Steve,
I think I made it! I just did one more PCR of PCR(i dont know if it makes sense) with the conditions that you have said. Thank you sooo much!!!
Kind regards,
Gunce
#2 Re: rf-cloning troubles » Problem with Secondary PCR » 2017-03-08 14:15:43
Dear Steve,
Firstly thank you so much for feedback and suggestions.
Unfortunately these changes did not work on my secondary PCR - I could not see any band on agarose.
I think I am losing my insert too much after the gel extraction- even if I use all the tricks about it, my insert ng is always around 2-3. (And my insert should be around 365 ng according to my project id)
By the way, here is my project id: http://www.rf-cloning.org/rf_cloning_project.php?proj_id=a0bf168a3f67aa0f4a5c6833dd351a11
I tried the extension time as the website said(without using primers and dropping the cycle number to 15), but again, I couldnt see any band on agarose. I would be so appreciated if you would have time to take a look at my project. I dont know what else I should do. 
Kind regards,
Gunce
#3 rf-cloning troubles » Problem with Secondary PCR » 2017-02-26 21:26:35
- gunception
- Replies: 7
Dear Steve,
First of all, I would like to thank you for this amazing tool and forum. 
I am having some problems at secondary PCR and finally decided to write here -where have I been! Anyway, my insert is 1524bps and I am using pET28TEV vector; and my new plasmid size should be 6784bps. (I had no problem with Primary PCR) So here is the conditions that I am using for Secondary PCR:
PCR Reaction mixture
•5 µl 10X KOD Hot Start DNA polimerase Buffer
•1.5 µl reverse primer (10 µM)
•1.5 µl forward primer (10 µM)
•1 µl KOD Hot Start DNA polimerase(1 U/µl)
•3 µl MgSO4 (25 mM)
•5 µl PCR product (extracted from gel, about 1.5 kbp)
•5 µl dNTP (2 mM)
•1 ul plasmid DNA pET28TEVCATPO
•27 µl sterile ddH2O, final volume 50 µl
1. initial denaturing at 94 C for 2 min (x1)
2.1 94 C for 35 sec (denaturing) ]
2.2 55-60 C for 30 sec (annealing) ] 30 cycle
2.3 68 C for 7 min (extension) ]
3. 10 min extension phase at 68 C (1x)
At first, I could not see any band on agarose, but when I do a second PCR from that product-using it as template, I saw a band at 7000 bps-with a big happy smile on my face- after that the steps are going like this: DpnI treatment, Transformation, Isolation, Treatment with Digestion Enzymes and Sequence analysing (every single clone in the plate, almost 15 clones). And then it turned out to be cloning was not successful- I was so close with one of those clones (sequence analysing and digestion say so), but it turned out to be there was a missing part of my insert.
Now I tried the same protocol above, but I could not get the same results; I cant even see a band. I was checking here http://www.rf-cloning.org/QandA.php, and I thought I should try 2-Step protocol. Also, I realized that I was also using the primers at Secondary PCR; do you think it could make more trouble for my PCR?
I would really appreciate any suggestions.
Thank you.
P.S: Sorry for my English.
Kind regards,
Gunce Goc
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