Cloning

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Sunil Abraham
Sunil Abraham's picture
Cloning

 I have designed expression primers without overhangs, I could able to amplify the DNA only with pfu polymerase and not with taq polymerase, now how can I do blunt end cloning, someone please help me 

Ruderus
Ruderus's picture
 You need to blunt-end ligate

 You need to blunt-end ligate your PCR product directly into you blunt-end vector... the ligation is inefficient, but can be done.
Make sure you follow these guidelines:

1) Dephosphorilate your vector after you linearize it, this is very important!!!!
2) use the minimum amount of vector possible... I use 25 ng per ligation.
3 Use a the insert in molar excess of 5:1 with respect to vector
4) Use regular T4 Ligase and let the ligation go O/N at 16 °C
5) Make sure you always do a control ligation without the insert... this should give zero colonies.

Also, if you get positive clones, you will need to verify that the insert is in the correct orientation, you can do that by digesting with restriction enzymes or by PCR, the choice is up to you.

I hope this helps, good luck!

Sunil Abraham
Sunil Abraham's picture
Thank u so much, I have

Thank u so much, I have produced the similar way many clones, i have confirmed it with colony PCR, R.E. digestion, sequencing and expressed too. I use double enzymes so I never dephosphorylate but I always use control and no colonies appear in control. I always design primers with overhangs eg: CACC or GACC in front of restriction sites so i m always successful in blunt end cloning.
But in this case its new for me the primers are without overhangs, I m not able to get a clone the vector is pET 5.8 kbp and the insert is 2589 kbp, I tried several times later i came to know the primer was designed by my junior on my absence for T.A cloning but I never got amplified by Taq polymerase and only by pfu polymerase only while checking i came to know no overhangs, bcoz earlier an year back my senior faced problem in cloning the similar way so he tried several ways always he got some mutation in T.A cloning so he tried pfu and not successful, by the suggestion of some experts he designed new primers with overhangs and he got clones successfully aft an year, so my doubt is that whether my condition is also similar to that or is there any chance for me to get a blunt end clone still without designing another primer with overhangs, please help me in this regard,
thank u.