Addition of 3' A overhangs to PCR products: Difference between revisions
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==Critical steps== | ==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== | ==Troubleshooting== | ||
Revision as of 21:00, 18 October 2008
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 
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
Acknowledgments
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References
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