Addition of 3' A overhangs to PCR products

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==Curators==
==Curators==
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Mohsen Hosseini
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[[User:S. Mohsen Hosseini|Mohsen Hosseini]]
==Abstract==
==Abstract==
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If PCR is performed using a proofreading DNA polymerase, such as ''Pfu'' DNA polymerase, the product will have blunt ends. ''Taq'' DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.
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If PCR is performed using a proofreading DNA polymerase, such as ''[[Pfu]]'' DNA polymerase, the product will have blunt ends. ''Taq'' DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.
==Materials==
==Materials==
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Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
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[[Category:Protocol]]
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[[Category:Protocol]][[Category:Needs attention]]

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Contents

Curators

Mohsen Hosseini

Abstract

If PCR is performed using a proofreading DNA polymerase, such as Pfu DNA polymerase, the product will have blunt ends. Taq DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.

Materials

Reagents

  • Purified PCR product
  • dATP
  • PCR buffer with Mg
  • Taq DNA Polymerase
  • ddH2O

Equipment

  • Thermocycler or Heat block equilibrated at 72°C
  • Spectrophotometer

Procedure

1. Purify the PCR product Image:Critical step.png

2. Prepare Taq DNA polymerase reaction mix for a typical 20 - 50 μl reaction:

Final Concentration Volume(μl)
Purified PCR product 0.15 to 1.5 pmol variable
dATP (10 mM) 0.2 mM 1
PCR Buffer with Mg (10x) 1x (1.5 mM MgCl2) 5
Taq DNA Polymerase (5 U/μl) 1U 0.2
ddH2O - up to 50 μl

The A-addition reaction works best when a specific amount of the PCR product is used. The recommended amount is 10–100 ng PCR product for each 100 bp length of the PCR product. This corresponds to 0.15–1.5 pmol PCR product (see table below).

PCR product size Amount of PCR product to use
100 bp 10–100 ng
250 bp 25–250 ng
1000 bp 100–1000 ng

3. Incubate 20 min at 72 °C.

4. Proceed to TA cloning. For optimal ligation efficiency, it's best to use fresh PCR products, since 3´A-overhangs will gradually be lost during storage.

Critical steps

Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.

Troubleshooting

Notes

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