Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
7 posts • Page 1 of 1
I want to ligate PCR product with T-vector, also I need high efficiency because the PCR product is random PCR. So, I only need a "A" at the PCR products ends to make sure the efficiency. How to do that.
I worried Taq would add poly A at the ends.
Thanks for the reply.
Actually, I am not sure it adds one or more A at the ends.
I just found in the manual of TOPO vector, "Another approach was to utilize the terminal transferase activity of Taq DNA polymerase which adds a single 3´-A overhang to each end of a PCR product. " It looks TAQ ONLY add one A.
Taq adds one A at the end. If you are using Pfu or another polymerase you might have some problem but If I remembre correctly by just making a mix of buffer (1x final), DNA and a few units of Taq and incubating a short time at 72C you can add the extra A at the end of a PCR product that did not had it in the first place. ALternatively some highfidelity mix are a mix of Taq and Pfu and have the A overhang too.
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
I find useful information but cannot post the link.
Taq does not always add an A to the 3' end of the PCR product.
(Hu, 1993 DNA and Cell Biology 12 (8) 763-770.,
D. Denney, Jr. and I. Weissman 1990 Amplifications 4: 25-26.,
V.L. Magnuson, D.S. Ally, S.J. Nylund, Z.E. Karanjawala, A.L. Lowe, S. Gough and F.S. Collins, 1996 Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications afor PCR-based genotyping and cloning. Nucleic Acids Res. 21:700-709.(see for a more detailed version of the chart below)
Hite, J., Eckert, K. A., Cheng K. C., 1996. Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)nd(G-T)n microsatelite repeats. Nucleic Acid Res. 24: 2429-2434.,
Costa and Weiner, 1994 NAR 22 (12) 2423.)
If the 3’ nucleotide of the PCR product strand is a T, then Taq modifies the 3’ end to - T, + A. (nucleotides shown in order of occurence)
If the 3’ nucleotide of the PCR product strand is a G, then Taq modifies the 3’ end to +G > + A > +C.
If the 3’ nucleotide of the PCR product strand is a C, then Taq modifies the 3’ end to + A > +C.
If the 3’ nucleotide of the PCR product strand is an A, then Taq modifies the 3’ end to + A.
This means that you need to design the 5' end of the primers to end in T in order to get an A added to the 3' end of the PCR product.
The number of PCR cycles affects the cloning efficiency when using TA-vectors (vectors that have a single TTP or UTP overhang designed to exploit the addition of a single 3’-dA to PCR products by Taq DNA polymerase. Performing excessive cycles that result in more template than enzyme may result in ragged ends due to incomplete elongation. Amplified PCR products (particularly from excess template) exist as a heterogeneous mixture of ragged ends, blunt ends and 3’-overhangs (extendase activity) and incompletely extended fragments that can generate PCR monsters during subsequent PCR cycles. The degree of heterogeneity is strongly influenced by the nucleotides immediately flanking the 3’-end.
The efficiency of a 3’-dA addition ranged from 4-75% depending on the nucleotide composition of the 3’-end of the PCR product.
( J. M. Brownstein, J. D. Carptena and J. R. Smith 1996 BioTechniques 20: 1004-1010.,
V. L. Magnuson, P. S. Ally, S. J. Nyland, Z. E. Karanjuwala, J. B. Rayman, J. I. Knapp, A. L. Lowe, S. Ghosh and F. S> Collins 1996 BioTechniques 21: 700-709.)
To promote “3’-dA” addition, an extended post-PCR incubation (10 to 90 minutes at 72C) is required and the base composition of the 3’-ends is restricted.
If you have used a proofreading polymerase (with 3′- 5′ exonuclease activity), then you will not have A overhangs capable of ligating to the T overhangs of your vector.
PCR reactions contain unincorporated dATP that is a competitive inhibitor for the ATP in the ligation reaction. 66 µM dATP in the ligation reaction will inhibit ligation 60%. (1992 BRL Technical Bulletin 5224-1. )
SOC gives 2x more transformants than LB. SOB/SOC contain twice the Bacto tryptone of LB and provide protein precursors for rapid repair of cell walls damaged by generation of competent cells and the electroporation process, maintain isotonicity to prevent cell death by osmotic rupture and SOC contains glucose for easy energy conversion. (R. Hopper 1995 Genetic Engineering News 15 (9) 18.)
There is now a listing on the OpenWetWare Wiki about adding 3' A overhangs using Taq polymerase.
The site listing can be found here: http://openwetware.org/wiki/Addition_of ... R_products
In order to overcome the fact that Taq will add non-A bases in some cases to the ends in a sequence dependent manner, you need to first purify your PCR product by using a PCR purification kit (I use the Qiagen column kit for PCR purification).
After purification, you can add ONLY buffer, template, dATP, and Taq. Then it's absolutely impossible for Taq to add non-A bases on the ends. Even if your efficiency is low, incorporation of the A will occur at a MUCH higher rate than if you used raw PCR product containing all 4 trinucleotides.
7 posts • Page 1 of 1
Who is online
Users browsing this forum: No registered users and 0 guests