Knight:Annealing and primer extension with Taq polymerase: Difference between revisions
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==Materials== | ==Materials== | ||
*Two oligos which overlap by ~20 bp | *Two oligos which overlap by ~20 bp. | ||
Oligo 1: 5' ----------------------------------- 3'<br> | Oligo 1: 5' ----------------------------------- 3'<br> | ||
Line 11: | Line 11: | ||
==Procedure== | ==Procedure== | ||
#Dilute the two oligos to a concentration of 25 μM using H<sub>2</sub>O | #Dilute the two oligos to a concentration of 25 μM using H<sub>2</sub>O. | ||
#*General information on [[Designing primers|primer design]]. | |||
#*Notes on [[Knight:Annealing and primer extension with Taq polymerase#Notes|ordering long primers]]. | |||
#*Notes on [[Knight:TOPO TA cloning#Notes|efficient addition of 3'A to PCR products]]. | |||
#Mix the following in a 0.6 mL sterile tube | #Mix the following in a 0.6 mL sterile tube | ||
#*9 μL PCR supermix | #*9 μL PCR supermix |
Revision as of 12:07, 26 May 2006
This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp) using Taq polymerase. The DNA fragment can be immediately used in a TA cloning reaction. (To proceed to a restriction digest step, purification is necessary.)
Materials
- Two oligos which overlap by ~20 bp.
Oligo 1: 5' ----------------------------------- 3'
Oligo 2: 3' ----------------------------------- 5'
Procedure
- Dilute the two oligos to a concentration of 25 μM using H2O.
- General information on primer design.
- Notes on ordering long primers.
- Notes on efficient addition of 3'A to PCR products.
- Mix the following in a 0.6 mL sterile tube
- 9 μL PCR supermix
- 0.5 μL oligo 1
- 0.5 μL oligo 2
- Anneal and extend the two oligos together by placing the mixture in a thermal cycler (MJ Research, PTC-200) and running the following protocol.
- 94°C for 5 mins
- 94°C for 30 seconds
- 56°C for 30 seconds (or whatever an appropriate annealing temperature is)
- 68°C for 30 seconds
- Repeat steps 2-4 2-3 cycles
- 68°C for 5 mins
- Use fresh 1μL PCR product in a TOPO TA cloning reaction.
Notes
- For oligos greater than 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. Invitrogen custom primers offers this service for an extra fee.