DNA Reaction - Long Distance PCR

Jeff Lawrence

1. Prepare Long PCR Buffer as:

	1x Buffer		5x Buffer
	85 mM	KOAc		4.25 mL	1 M KOAc
	25 mM	Tricine, pH 8.7	1.25 mL	1 M Tricine, pH 8.7 @ 25° with KOH
	8 %	Glycerol	4.00 mL	Glycerol
	1 %	DMSO		0.50 mL	DMSO

2. Select PCR primers of 20 to 23 bases in length, G+C of 12 bases, A+T of 8 - 11 bases. Ideal Tm of 60° to 68° in 85 mM salts.

3. Mix the following for amplification from plasmids of bacteriophages:

	1.0 µL	10-2 Dilution of Bacteriophage DNA 
	10.0 µL	5x Long PCR buffer
	5.0 µL	8 mM dNTP (2 mM each)
	5.0 µL	5 µM Primer 1
	5.0 µL	5 µM Primer 2
	5.0 µL	11 mM Mg(OAc)2
	0.3 µL	Tth DNA Polymerase, at 5 unit/µL
	0.1 µL	Vent DNA polymerase, at 0.2 units/µL
	18.6 µL	ddH₂O						
	50.0 µL	Total volume

4. Mix the following for amplification from bacterial DNA:

	1.0 µL	10-2 Dilution of Bacterial DNA 
	10.0 µL	5x Long PCR buffer
	5.0 µL	8 mM dNTP (2 mM each)
	5.0 µL	5 µM Primer 1
	5.0 µL 	5 µM Primer 2
	5.0 µL	11 mM Mg(OAc)2
	0.3 µL	Tth DNA Polymerase, at 5 unit/µL
	0.5 µL	Vent DNA polymerase, at 0.2 units/µL
	18.2 µL	ddH₂O						
	20.0 µL	Total volume

5. DO NOT perform Long Distance PCR in Glass capillary tubes; Tth Polymerase does not like glass. Perform in plastic. If you must do it in glass, be sure to add BSA.

6. Find conditions for PCR by varying this base program:

	Initial Melting : 94° for 10 - 15 sec
	Cycles 1 - 15 : 94° for 10 sec, 68° for (1 min + 25 sec/kb)
	Cycles 15 - 30 : 94° for 10 sec, 68° for (1 min + 25 sec/kb + 15 sec/cycle)

Last Update: Thursday, 12-Dec-2024 00:55:25 UTC

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Eric Kofoid eckofoid at ucdavis.edu