Knight:TOPO TA cloning

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Notes)
Line 1: Line 1:
 +
==Purpose==
 +
 +
Efficient cloning of PCR products.
 +
 +
==Materials==
 +
*PCR product generated with Taq polymerase (for 3' A tails)
 +
*[http://www.invitrogen.com/content.cfm?pageid=4073 TOPO cloning kit]
 +
 +
==Procedure==
 +
===Reshma's procedure===
 +
(Note that the kit details a slightly different procedure which may give superior efficiencies.)
 +
#Mix 1 μL PCR product (amplified with [http://www.invitrogen.com/content/sfs/manuals/11306016.pdf Platinum PCR supermix]) with 0.5 or 1μL TOPO vector (0.5μL should be sufficient.)
 +
#Incubate 5 mins on the bench top
 +
#Place on ice
 +
#Transform
 +
==Notes==
==Notes==
*[[Tom Knight]] claims you can cut the reaction volume in half and it will still work fine.
*[[Tom Knight]] claims you can cut the reaction volume in half and it will still work fine.
*Use fresh PCR product for best results.  (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
*Use fresh PCR product for best results.  (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
*Your primers should NOT have 5' phosphates.  They interfere with topoisomerase I.
*Your primers should NOT have 5' phosphates.  They interfere with topoisomerase I.
-
 
+
*From [[Tom Knight]]: As this [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8780871&query_hl=4 article] discusses, the 5' end of the PCR primer  has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR.  Since it is often necessary to add such an overhang to PCR products for cutting of an added cloning site, and since for such applications the sequence is immaterial, we should probably standardize that sequence to the A tail favoring sequence, GTTTCT for all PCR primers we make.  This will favor a 3' A overhang on the PCR products, and allow TA cloning, or TOPO TA cloning of these products.  Blunt end cloning could still be done by using Pfu or Phusion enzymes.  I can't make any sense from the result about extending the primer sequence another base which is not specified.  It seems to me this can not have any effect.
 +
 
==Resources==
==Resources==
*[http://www.invitrogen.com/content.cfm?pageid=4073&CFID=2390357&CFTOKEN=58806917 Website]
*[http://www.invitrogen.com/content.cfm?pageid=4073&CFID=2390357&CFTOKEN=58806917 Website]
Line 10: Line 27:
*[http://www.invitrogen.com/content/sfs/brochures/710_021849%20_B_TOPOCloning_bro.pdf Brochure]
*[http://www.invitrogen.com/content/sfs/brochures/710_021849%20_B_TOPOCloning_bro.pdf Brochure]
*[http://www.invitrogen.com/search.cfm?category=Vectors&searchTerm=pCR4%2DTOPO&id=243 pCR4-TOPO]
*[http://www.invitrogen.com/search.cfm?category=Vectors&searchTerm=pCR4%2DTOPO&id=243 pCR4-TOPO]
 +
 +
==References==
 +
 +
M. J. Brownstein, J. D. Carpten, and J. R. Smith. Modulation of non-templated nucleotide addition by taq dna polymerase: primer modifications that facilitate genotyping. Biotechniques, 20(6):1004–6, 1008–10, 1996.

Revision as of 14:49, 30 September 2005

Contents

Purpose

Efficient cloning of PCR products.

Materials

Procedure

Reshma's procedure

(Note that the kit details a slightly different procedure which may give superior efficiencies.)

  1. Mix 1 μL PCR product (amplified with Platinum PCR supermix) with 0.5 or 1μL TOPO vector (0.5μL should be sufficient.)
  2. Incubate 5 mins on the bench top
  3. Place on ice
  4. Transform

Notes

  • Tom Knight claims you can cut the reaction volume in half and it will still work fine.
  • Use fresh PCR product for best results. (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
  • Your primers should NOT have 5' phosphates. They interfere with topoisomerase I.
  • From Tom Knight: As this article discusses, the 5' end of the PCR primer has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR. Since it is often necessary to add such an overhang to PCR products for cutting of an added cloning site, and since for such applications the sequence is immaterial, we should probably standardize that sequence to the A tail favoring sequence, GTTTCT for all PCR primers we make. This will favor a 3' A overhang on the PCR products, and allow TA cloning, or TOPO TA cloning of these products. Blunt end cloning could still be done by using Pfu or Phusion enzymes. I can't make any sense from the result about extending the primer sequence another base which is not specified. It seems to me this can not have any effect.

Resources

References

M. J. Brownstein, J. D. Carpten, and J. R. Smith. Modulation of non-templated nucleotide addition by taq dna polymerase: primer modifications that facilitate genotyping. Biotechniques, 20(6):1004–6, 1008–10, 1996.

Personal tools