RF-Cloning.org Forum

gunception

Member

From: Turkey

Registered: 2017-02-26

Posts: 3

Problem with Secondary PCR

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

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Problem with Secondary PCR

Hi Gunce,
I recommend that you make two small changes; remove the fwd and rev primers from the secondary reaction and drop the number of cycles in the secondary to 15.
Give it another go, and let us know how it turns out!
Best of luck,
-Steve

gunception

Member

From: Turkey

Registered: 2017-02-26

Posts: 3

Re: Problem with Secondary PCR

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

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Problem with Secondary PCR

Hi Gunce,
Sorry to hear you're having trouble. The project looks fine to me, so I'm guessing that the gel extraction is the primary issue. Are you getting nice juicy bands from the 1° PCR? Can you switch to a different kit?
Other things that may help:
   — The Tm on the plasmid sides of your primers is pretty high, so maybe try an annealing temp of 63
   — The elongation rate on KOD is >100 bases/sec, so your extension time of 7 mins is overkill. Try the ~2min recommended by the site. (edit: oops, I just saw that you tried this already)
-Steve

ps. I wouldn't get too worried over what the 2° looks like on a gel. You can have a successful reaction below detection level, so I pretty much gave up on visualizing the 2° PCR.

gunception

Member

From: Turkey

Registered: 2017-02-26

Posts: 3

Re: Problem with Secondary PCR

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

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Problem with Secondary PCR

Great! I assume you haven't sent it off for sequencing yet, so stay cautiously optimistic
I hope it comes back clean for you.
Best,
-Steve

ChesterMo

Member

Registered: 2017-11-24

Posts: 1

Re: Problem with Secondary PCR

Steve, can you please explain what exactly made the difference here?

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Problem with Secondary PCR

Hi Chester,
I made four recommendations here:

1)  Remove fwd and rev primer from 2°
        -> These primers are for creating the mega primer only, and will compete/interfere in the 2°
2) Reduce 2° cycles to 15
        -> High molecular weight products start to accumulate with increased cycle number (see Bryksin et. al., 2010)
3) Increase annealing temp to 63°C
        -> A higher annealing temp reduces mis-pairing and extension of spurious product
4) Reduce extension time
        -> Polymerase degrades over time at temperature, so leaving the reaction at 68°C for 3X longer than necessary (given the processivity of the enzyme) is counter productive.

I'm not exactly sure what Günce meant by 'PCR of PCR', so if this was the secret sauce you might need to reach out to her directly. Please let us know if you do!
Best,
-Steve