Annealing primers

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 100^oC 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.

Materials

• 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'[math]\displaystyle{ \rightarrow }[/math]5' exo^- polymerase
• dNTPs (25 mM each dNTP in stock)
• Restriction enzyme(s)
• Restriction enzyme buffer
• BSA

Calculating amount of oligo for reaction

This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)

[math]\displaystyle{ \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock} }[/math]

Procedure

1. Dilute the two oligos to a concentration of 10 or 25 μM using H₂O
2. 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 1 (typically 1 μg or more)
   • (87 - 2X) μL deionized sterile H₂O
3. Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94°C for 5 mins followed by a cool down for 0.1°C/sec to 37°C or by placing the tube in a beaker of boiling water and letting it cool to room temperature.
4. Add 1 μL Klenow 3'[math]\displaystyle{ \rightarrow }[/math]5' exo^- polymerase to mixture.
   Vortex polymerase before pipetting to ensure it is well-mixed.
5. Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP)
   Recommend using a thermal cycler for the following incubation steps.
6. Incubate 1 hr at 37°C.
7. Heat inactivate polymerase by incubating at 75°C for 20 minutes.
   See Restriction Digest for more information on the following steps.
8. Add 1 μL restriction enzyme(s) to mixture.
9. Incubate for a minimum of 2 hrs.
10. Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
11. Purify DNA as necessary