Knight:Annealing and primer extension with Taq polymerase

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'

• PCR supermix

Procedure

1. 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.
2. Mix the following in a 0.6 mL sterile tube
   • 9 μL PCR supermix
   • 0.5 μL oligo 1
   • 0.5 μL oligo 2
3. Anneal and extend the two oligos together by placing the mixture in a thermal cycler (MJ Research, PTC-200) and running the following protocol.
   1. 94°C for 5 mins
   2. 94°C for 30 seconds
   3. 56°C for 30 seconds (or whatever an appropriate annealing temperature is)
   4. 68°C for 30 seconds
   5. Repeat steps 2-4 2-3 cycles
   6. 68°C for 5 mins
4. 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.
• RS 21:30, 14 June 2006 (EDT): I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens.
• Reshma 19:49, 19 October 2006 (EDT): Tom suggests considering how much enzyme you use relative to how much primer. Supposedly, polymerase does not necessarily fall off the ends of the DNA which means in 2-3 cycles, very few of your annealed primers will become completely double-stranded (as this requires two polymerases to bind to the same annealed primer molecule and extend). This lack of complete double stranding could increase the potential for errors.

References

1. Stemmer WP, Crameri A, Ha KD, Brennan TM, and Heyneker HL. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995 Oct 16;164(1):49-53. DOI:10.1016/0378-1119(95)00511-4 |