adding resistriction ezymes to primers - cloning (Jul/19/2006 )

Hi,

I am trying to clone a 3 kb fragment into the pGL 3.0 basis vector but the cloning has not been successful. I am able to get the pcr product when I use primers without res. enzy for amplification by when I add the res. enz seq on either side then I am not getting the product. So I tried out couple of enzymes like the LA ken Taq from sigma and the Phusion but again I getting the product but not able to clone.

Any suggestions or solution will be appreciated.

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U should change PCR conditions for the primers with the restriction sites.

And since u got the PCR product (one with restriction sites) with other enzymes, u should try different digestion times for the PCR (shorter).

Which enzymes r u digesting with and for how long?

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some RE require extra bases around in order to recognize and cut the DNA. so we need to add extra bases other than enzyme recognition seq at the 5' of the primer. u may check that in NEB site:

http://www.neb.com/nebecomm/tech_reference...ized_vector.asp

if u have the fragment of desired lenght after PCR, then u may not be digesting your piece well.

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hi

i am using NheI& hindIII, XhoI & HindIII.

Digesting the PCR product with .5 ul of enzyme directly for 12h or over night at 37c.

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antimatter -

wow, I think that's a long time to do the digestion; have you tried it shorter? I would be concerned about star activity, even with such small amount of enzyme...perhaps use more enzyme for a shorter time?

also, please post specifics about your PCR protocol, and your primers, and their Tm's, etc...I suspect the problem lies with your PCR and fiddling too much with downstream steps will just cause you undue frustration without solving the problem

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hi

pcr : 98 for 1 min, 98 for 10s, annealing temp 30s and 72 for 15s (1000bp) 30 cycles. The annealing temp varies and I calculate it from the finnzyme webpage as I am using the phusion taq pol from finnzymes.

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antimatter, have you adjusted your PCR protocol to allow for the addition of bases to your primers?

for example, start the PCR for 10 cycles at the Ta of the primers if they didn't have RE sites added...calculate the Ta by using only the parts that align with your template

then, after the 10 cycles, increase the Ta to account for the entire primer

do you see? or did you already know this?

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Wow. Is this true? and does this work?

When I have tried PCR with primers that have extra sequence corresponding to the RE site and the additional flanking 4-6bases, I have always kept the annealing temperature based on the Tm of the region that aligns (and not the entire primer) for the entire 30 cycles.

And I never get a PCR product!!

Should I use a lower TM (corresponding to the region that aligns) for the first 10 cycles.
Then stop the reaction.
Start the PCR again with a higher annealing temperature corresponding to the higher Tm of the entire primer.

Is this correct??

Please let me know.
Thanks

[

quote name='aimikins' date='Jul 28 2006, 10:51 AM' post='61647']
antimatter, have you adjusted your PCR protocol to allow for the addition of bases to your primers?

for example, start the PCR for 10 cycles at the Ta of the primers if they didn't have RE sites added...calculate the Ta by using only the parts that align with your template

then, after the 10 cycles, increase the Ta to account for the entire primer

do you see? or did you already know this?
[/quote]

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Adding the RE cut site (and an overhang) to working primers sometimes creates hairpins and primer-dimers not present in the original (working) primers. Check you complete primer sequence for hairpins and dimers at the IDT site (www.idtdna.com). If you are getting product and are not able to clone, then the problem is likely (1) inadequate overhang at the 5' end of the primer or (2) inadequate cleanup of the PCR enzyme prior to digestion.

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