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#1 2014-05-29 14:49:37

Tamjeed
Member
Registered: 2014-05-29
Posts: 3

Suggestion for Primer design and conditions

I have used RF-cloning successfully several times.
Recently I have attempted to substitute 8 aa using the technique. I have made two sets of primers that are slightly different. Both sets have not worked. I have also eliminated the 55C annealing temperature and gone on with the 2 step protocol (68C annealing and elongation).
My two project files are below

http://www.rf-cloning.org/rf_cloning_pr … becb9fc7a4

http://www.rf-cloning.org/rf_cloning_pr … c1a0bcb569

Is this because of poor primer design?

Thank you
Sincerely,
Tamjeed Saleh

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#2 2014-05-29 15:23:27

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Suggestion for Primer design and conditions

Hey Tamjeed,
Are your plasmids really that tiny? If you've only included a portion of the destination plasmid, make sure you are running the extension step for an appropriate amount of time. The primers themselves seem to be a bit unbalanced in terms of their annealing temps, which you should probably try to get within a 1-4°C of one another, but they don't seem to suffer from terrible self-priming at least.
What were your exact PCR conditions (both reaction mix and cycling)?
-Steve

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#3 2014-05-29 16:16:08

Tamjeed
Member
Registered: 2014-05-29
Posts: 3

Re: Suggestion for Primer design and conditions

Hello Steve,
Thank you for your reply.
The plasmid I am using is a pet42a backbone with my 255 a .a. gene already inserted; so total approx 8kb. The gene was inserted between the Nco1 site and the Ase1 site using RF cloning. (YAH!! that time it worked beautifully!)
The PCR mix was 80ng of plasmid, 300ng of primer,
PCR cycle   95C   2min
                 Then 18 cycles of
                 95C for 30 seconds
                 68C for 9 minutes
then a final elongation stem of 68C for 12 minutes.

I am using Pfu ultra. I use 1.5ul DMSO in the mix. the reaction volume is 50ul.

I don't use the full backbone because its just convenient to enter sequence I get from sequencing the constructs. I have had success subcloning larger fragments before. In this case I want to insert a series of Prolines; maybe that is giving me issues.

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#4 2014-05-29 16:34:00

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Suggestion for Primer design and conditions

Cool, I think there is hope. Give the reaction another try with the following modifications:
- Set up the mix without any plasmid to start, and reduce the amount of each primer to 5 ng (300 ng is over-kill, and could be a problem)
- Run a 5 cycle PCR (hang out by the machine until it's done, so it isn't sitting around)

      1 - 95°C 2 min
      2 - 95°C 30 sec
      3 - 56°C 30 sec
      4 - 68°C 30 sec    GOTO 2 4x
      6 - 20°C hold

- Squirt in your 80 ng of plasmid
- Execute the full 18 cycle PCR
- Cross your fingers

Let us know how it turns out!
-Steve

ps. I doubt the prolines are an issue.

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#5 2014-06-03 16:57:44

Tamjeed
Member
Registered: 2014-05-29
Posts: 3

Re: Suggestion for Primer design and conditions

Hi Steve,
It worked beatifully!
Thank you very much for the advice.

Sincerely,
Tamjeed

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#6 2014-06-03 16:59:52

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Suggestion for Primer design and conditions

That's great! Congrats.

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