Annealing complementary primers
A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as Invitrogen.
- When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl.
- For the annealing mix one recipe that works is as follows -
- 4 μL of each of the concentrated primers.
- 4 μL of salt solution (10 mM NaCl)
- 28 μL of water
- The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
- Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a Thermocycler. Melting temperature calculations can best be done using software such as VectorNTI or data may come with the primers themselves.
- A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100oC and certainly any non-specific binding should be melted at that temperature.
- Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as PNK Treatment of DNA Ends
Annealing and primer extension
This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.
- Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below. See Restriction Digest for notes on cutting near the ends of linear DNA fragments.
Oligo 1: 5' ---RE site-------------------------------- 3'
Oligo 2: 3' --------------------------------RE site--- 5'
- Klenow 3'5' exo- polymerase
- dNTPs (25 mM each dNTP in stock)
- Restriction enzyme(s)
- Restriction enzyme buffer
Calculating amount of oligo for reaction
This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)
- Dilute the two oligos to a concentration of 10 or 25 μM using H2O
- Mix the following in a 0.6 mL sterile tube
- 10 μL 10X restriction enzyme buffer
- 1 μL 100X BSA
- X μL oligo 1 (typically 1 μg or more)
- Y μL oligo 2 (typically 1 μg or more)
- (87 - X - Y) μL deionized sterile H2O
- Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
- Add 1 μL Klenow 3'5' exo- polymerase to mixture.
Vortex polymerase before pipetting to ensure it is well-mixed.
- Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP).
Recommend using a thermal cycler for the following incubation steps.
- Incubate 1 hr at 37°C.
- Heat inactivate polymerase by incubating at 75°C for 20 minutes.
See Restriction Digest for more information on the following steps.
- Add 1 μL restriction enzyme(s) to mixture.
- Incubate for a minimum of 2 hrs.
- Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
- Purify DNA as necessary
W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. PubMed