LM-PCR worksheet.  Modified 7/19/02 to correct for precipitate problems in the Ligation dilution solution and the ligation premix and for use of the DNA engine thermocycler.  Recipes for stock solutions marked with * are found here.  The tables are set up to aid in making master mixes.  For information of commonly used primer sets, go here.  Modification 10/9/06: Song Tan's Pfu polymerase can be substituted for Vent polymerase.  Song Tan's T4 DNA ligase can be substituted for the USB ligase.  Song's ligase is very concentrated so approximately 0.01 should be used per reaction (ie, 0.1 ul of ligase for 10 reactions!)

Description of experiment:
 
 
 

Combine 10 ul of DNA (100 ng in 0.5X TE) with 20 ul of First strand synthesis mix. (Earlier protocols had the DNA in 5ul of TE.  I've increased the volume to help dissolve the DNA.)
 ____X
 First strand synthesis mix (20ul/rxn)
 _____
 12.75 ul H2O
 _____
 6ul 5X First Strand Buffer* (warm at 37°C to dissolve gelatin)
 _____
 0.5 ul of 1 pmoles/ul first primer (increased per Hongbing's recommendation)
 _____
 0.5 ul 10 mM dNTPs
 _____
 0.25 ul 2U/ul Vent polymerase (NEB #254L)

In the thermocycler, subject the samples to the following temperatures.
 
first primer
 Thermocycler notes:
Denaturation 5'
95°C
Annealing 30'
_____
Extension 10'
76°C
Storage 10°C

Spin down condensate and place on ice.

Add 17.5 ul of Modified Ligation Dilution Solution* and 2.5 ul of 1 mg/ml of BSA to each sample (BSA and MLDS can be premixed just before use, prepare 2 samples worth more than needed to insure that there is enough.)

Add 25 ul of Ligation mix.
____X
 Ligation Mix (25ul/rxn)
_____
 9.25 ul Modified Ligation Premix*
_____ 1.25 ul 1mg/ml BSA
_____
 7.5 ul 10 mM rATP
_____
 5 ul Linker pair (A':B at 20 pmoles/ul*)
_____
 2 ul T4 DNA ligase (1Weiss Unit/ul, low dilution from USB) or 0.01 ul of Song Tan's ligase (make up difference in volume using water)

Incubate overnight at 15°C in the Thermocycler.

After completing the ligations, add 9.4 ul of salt precipitation mix* and 220 ul cold Ethanol. Mix well and place on ice for at least 15 minutes. Microfuge in cold for 20 minutes. Wash one time with 100 ul of 75% ethanol, microfuging to secure the precipitate. Air dry the pellet.

Dissolve samples in 30 ul of H2O. Place sample on ice.

Add 70 ul of Vent amplification buffer.
____X Vent Amplification buffer (70ul/rxn)
_____
 45.5 ul H2O
_____ 20 ul 5X Vent Amplification Buffer* (warm @ 37°C to dissolve gelatin)
_____ 1 ul of 10 pmole/ul 2nd primer
_____ 1 ul of 10 pmole/ul Linker primer A'
_____ 2 ul 10 mM dNTP
_____ 0.5 ul Vent polymerase

Perform 18 amplification cycles. The first cycle should have a 3 minute denaturing step at 95°C and the remaining denaturation steps should be for 1 minute. All cycles have annealing steps of 2 minutes. The extension time begins with 3 minutes and is increased by 5 seconds with each cycle.  All extension steps are at 76°C. Following amplification, the samples are stored 10 degrees.
 
2nd primer
 Thermocycler notes:
Denaturation
95°C
Annealing
______
Extension
76°C
Storage 10°C

If it is not already done, radiolabel the third primer with kinase. The following reaction will give you enough primer to label 20 samples if you adjust the final volume of the primer so that it is a little more than 100 ul.
 
____X
 Primer-labelling for 20 samples
_____ H2O so the final reaction volume is 10 ul
_____ 1 ul of 10X kinase buffer
_____ 100 pmoles of 3rd primer
_____ 0.2 ul kinase
_____ 2 ul 32P-gATP (6000Ci/mmole; 150uCi/ul, add last to minimize contamination)

Incubate kinase reaction at 37°C for 1 hour. Purify primer with a Bio-Rad Micro Bio-Spin 6 column according to Bio Rad's instructions.  The Bio-Spin 6 columns are spun at 1000 x g, which corresponds to a setting of 3.5 (3500 rpm) on the 5415C centrifuge.  The kinased sample should be diluted to 50 ul with 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA (STE) before applying to the column.  You'll recover radioactive DNA in a volume of 60 to 100 ul.  The  primer concentration is about 1 pmole/ul at 2 to 4 x 106 cpm/ul.

After the 18 cycles of amplification, proceed with the labelling reaction. Add 20 ul of labelling mix to each sample.
 
____X Labelling mix (20ul/rxn )
_____ 9.5 ul H2O
_____ 4 ul 5X Vent Amplification Buffer* (dissolve gelatin)
_____ 1 ul 10 mM dNTP
_____ 5 ul radiolabelled 3rd primer (approximately 2 pmoles) - I've had success increasing the volume of label to 10 ul and reducing water accordingly.
_____ 0.5 ul Vent Polymerase

Perform two cycles of labelling. The first cycle is initiated with a 3 minute denaturation at 95°C and the second cycle is for 1 minute. Annealing is done for 2 minutes. Extensions are done for 10 minutes at 76°C.
 

3rd primer Thermocycler notes:
Denaturation 95°C
Annealing ______
Extension 76°C

Following the labelling cycles, hold tubes at room temperature.

At the end of the labelling reaction, add 14 ul of Stop solution*.  Transfer 120 ul of each reaction to a fresh 1.7 ml tube (the larger tube facilitates the organic extraction).  Extract one time with 120 ul of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) {Caution, highly radioactive!). Transfer 100 ul of the aqueous phase to a fresh 1.7 ml tube and add 300 ul of cold Ethanol. Chill on ice for 15 minutes and then microfuge for 15 minutes. Remove supernatant and wash pellet with 100 ul of 75% ethanol, microfuge and discard supernatant. Air dry the samples.

Dissolve precipitates in 10 to 15 ul of sequencing loading buffer. For gel analysis, boil 3-5 ul and reserve the rest for a subsequent gel if this is needed.