LMPCR stock solutions

LMPCR Stock solutions (updated 10/9/06 with alternative preparation of A':B linker pair):
5X First Strand Buffer (Store in freezer, warm to 37^oC before use to thoroughly dissolve gelatin)

450 ul H₂O
200 ul 1M NaCl
50 ul 1 M Tris-Cl, pH 8.9
250 ul 100 MgSO₄
50 ul 1% gelatin

Modified Ligation Dilution Solution (MLDS, store in freezer)

655 ul H₂O
110 ul 1M Tris-Cl, pH 7.5
60 ul 300 mM MgCl₂
50 ul 1 M DTT

Modified Ligation premix (Store in freezer)

792 ul H₂O
83 ul 300 mM MgCl₂
50 ul 1 M DTT

Salt Precipitation Mix (Store in freezer)

840 ul 3 M Sodium acetate, pH 7
100 ul 10 mg/ml yeast tRNA

5X Vent Amplification Buffer (Store in freezer, warm to 37^oC before use to thoroughly dissolve gelatin)

200 ul 1M NaCl
100 ul 1 M Tris-Cl, pH 8.9
250 ul 100 mM MgSO₄
50 ul 1% gelatin
50 ul 10 % Triton X-100
350 ul H₂O

Stop Solution (Store in freezer)

100 ul 0.5 M Na₂EDTA pH 8.0
600 ul 3 M NaOAc pH 7.0

Preparation of A':B linker pair

In a 1.5 ml microfuge tube, prepare 1 ml of solution containing final concentrations of 250 mM Tris-Cl pH 7.7, 20 uM Linker A' and 20 uM Linker B. Heat the solution to 95^oC for 5 minutes and then float the tube in a beaker containing 250 ml of water adjusted to 70^oC. Allow the water to cool to room temperature and then leave the tubes at room temperature for at least one more hour. The beaker of water containing the tube of oligonucleotides is then place on ice so that it slowly cools to 4^oC and then the tubes are left on ice overnight. Store the oligonucleotide solution at -20^oC. Don't warm the solution above room temperature when you thaw the tubes.

Alternatively, the oligonucleotides can be annealed in the PCR machine. Divide the solution above into 5 x 200 ul portions in PCR tubes. Subject to the following: 95^oC for 5', 70^oC for 5', 65^oC for 5' , continue decreasing 5^oC increments and incubating 5' until the samples reach 20^oC, 20^oC for 60', 18^oC for 5', 16^oC for 5', 14^oC for 5', 12^oC for 5', 10^oC until its convenient to put at 4^oC overnight. Finally, store in the freezer.