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Problem with TA PCR cloning - no insert in white colonies - (Mar/22/2005 )

Dear colleagues:

I have a problem with T/A PCR cloning:

1) I am trying to clone four inserts of the following sizes: 498 bp, 510 bp, 910 bp and 1510 bp. I amplified all of them with Pfu+Taq (Fermentas)

2) I checked the amplification products by agarose gel, confirming sharp, clear products and a great yield.

3) Then I purified the bands with Rapid Gel Extraction Kit (Marligen Bioscience), and checked the purification by agarose gel , where I had pure bands of the expected size.

4) Then I incubated the gel-purified PCR products with Taq and ATP just in case the terminal A┤s were lost.

5) Then I used the product to ligate into a pGEM-T Easy (Promega), did an overnight ligation , using the manufacture┤s recomendations.

5) Then I precipitated the DNA with ethanol and acetate and resuspended in ddwater.

6) I used an electroporator to do the transformation , using XL1 electrocompetent cells and 1 uL of the resuspended ligation.

7) Plaqued the cells on LB/Amp (100 ug/mL), X-Gal and IPTG

8) I got lots of white colonies and few blue ones. Everything seemed fine. I did a colony-PCR to check on a few of the white colonies but they were all negative for the insert (the PCR was done using both positive and negative controls)

9) Then , I did an alkaline miniprep (just to use a different method for checking the presence of the insert) and digested with Eco RI, but no insert was released!

Ive done 20+ Colony-PCRs and some digestions of all white colonies (and some blue ones too!) but none of them have the insert!!! How come?
Has this ever happened to you? Im just starting my postgrad so I dont have much experience....some help would be so welcomed!
Thanks for your time....

Mathias
(Montevideo, Uruguay)

-matiascs-

QUOTE (matiascs @ Mar 22 2005, 01:09 PM)
1) I am trying to clone four inserts of the following sizes: 498 bp, 510 bp, 910 bp and 1510 bp.  I amplified all of them with Pfu+Taq (Fermentas)


Hi Mathias,

I Pfu has proofreading ability and will probably not leave an A overhang. if you do not require high fidelity template use normal taq instead.

That's why you are probably not getting your insert in!

Good luck!

Nick

-methylnick-

TA cloning requires ovehanging A, which is added to the amplicon oly by taq polymerase. Pfu is a proof reading enzyme and does produces blunt ended amplicons. So, either change to taq or go for blunt end cloning of Pfu ampified sample.

-sharath-

Hi Mathias,

Using the mix of Pfu and Taq should produce an A overhang. The Pfu has 3' to 5' exonuclease activity and no extendase activity, whereas the Taq should add the 3' terminal A. The only problems I see with your protocol are:
1/ do you clean up your DNA product after your additional Taq step (I don't think this Taq step is really needed) and visualise the product on a gel before ligating to check your end concentration. Every time you purify you lose a significant amount of DNA.

2/ If you do not purify I would never put more than 10% total ligation volume from your PCR reaction. The salt in the PCR reaction will interfere with the ligation efficiency.

Therefore there is two things to check insert concentration and salt concentration in ligation. However neither suggestion explains why you are getting white colonies which suggests something is going in which again leads me to think that the additional Taq step without clean up afterwards may be causing the problem. If it was me I would omit that step as your original mix should be good or just change to Pfu and blunt the inserts into a vector.

Hope this helps

Scott

-Scott-

Dear Scott,

Thanks for your reply...

About the "Taq Step": I did it because I tried before to ligate directly from an amplification with Pfu+Taq and got no colonies...adding this step gave me well over 1K white colonies...

I think you are right about the fact that I didnt clean out the Taq Step...I will perform a Phenol/Chloroform step to clean things out....I read in a paper that they didnt do it and work..whatever...

Also, my mentor suggested to try different insert/vector ratios and also to heat the mix with insert and vector (to 65║C) before putting the ligase (he said it would "open" the molecules)...

Thanks again to all who got interested...

Mathias
Montevideo, Uruguay



QUOTE (Scott @ Mar 23 2005, 06:31 AM)
Hi Mathias,

Using the mix of Pfu and Taq should produce an A overhang.  The Pfu has 3' to 5' exonuclease activity and no extendase activity, whereas the Taq should add the 3' terminal A.  The only problems I see with your protocol are:
1/ do you clean up your DNA product after your additional Taq step (I don't think this Taq step is really needed) and visualise the product on a gel before ligating to check your end concentration.  Every time you purify you lose a significant amount of DNA.

2/ If you do not purify I would never put more than 10% total ligation volume from your PCR reaction.  The salt in the PCR reaction will interfere with the ligation efficiency.

Therefore there is two things to check insert concentration and salt concentration in ligation.  However neither suggestion explains why you are getting white colonies which suggests something is going in which again leads me to think that the additional Taq step without clean up afterwards may be causing the problem. If it was me I would omit that step as your original mix should be good or just change to Pfu and blunt the inserts into a vector.

Hope this helps

Scott

-matiascs-

Hi Mathias,

As for the false positives in the ligation the only thing I could suggest is that for some reason the extra Taq step is giving you some abortive short fragments that ligate in to create the white colonies. Cleaning up after this step should get rid of any short products and hopefully eliminate false positives.

I also think your mentor is right try different ratios of insert:vector even up to 10:1, it should help to really drive the ligation.

If you have no luck I would still try the ligation straight from the Pfu/Taq with an increased insert:vector ratio.

Good Luck

Scott

-Scott-

Thanks again Scott, Ill try it out...
Mathias



QUOTE (Scott @ Mar 24 2005, 02:35 AM)
Hi Mathias,

As for the false positives in the ligation the only thing I could suggest is that for some reason the extra Taq step is giving you some abortive short fragments that ligate in to create the white colonies.  Cleaning up after this step should get rid of any short products and hopefully eliminate false positives.

I also think your mentor is right try different ratios of insert:vector even up to 10:1, it should help to really drive the ligation.

If you have no luck I would still try the ligation straight from the Pfu/Taq with an increased insert:vector ratio.

Good Luck

Scott

-matiascs-

Hi,


In your place,

- I only use classical Taq polymerase (if you don't want to express protien later) which adds A to your amplicon to overhang with T in the pGEM-T vector.
- I use more than 1Ál of resuspended ligation reaction : in my case, I resuspend the precipitated vector in 15 Ál and use 7-8Ál for the cloning. it works well.



good luck






QUOTE (matiascs @ Mar 25 2005, 10:53 AM)
Thanks again Scott, Ill try it out...
Mathias



QUOTE (Scott @ Mar 24 2005, 02:35 AM)
Hi Mathias,

As for the false positives in the ligation the only thing I could suggest is that for some reason the extra Taq step is giving you some abortive short fragments that ligate in to create the white colonies.á Cleaning up after this step should get rid of any short products and hopefully eliminate false positives.

I also think your mentor is right try different ratios of insert:vector even up to 10:1, it should help to really drive the ligation.

If you have no luck I would still try the ligation straight from the Pfu/Taq with an increased insert:vector ratio.

Good Luck

Scott


-MPK-