Polymerase Chain Reaction Protocol

Overview

This is a standard PCR protocol used on all first pass (unoptimized) PCR amplifications. This protocol outlines:

Setup of a single PCR reaction
Preparation of PCR-ready 96-well plates with Elongase MasterMix
PCR setup for amplification

We have tested numerous PCR reagent kits from different vendors (e.g., AmpliTaq (Perkin-Elmer)) to identify a kit which produces simple, robust, and very specific reactions, and allows for most PCR reactions to be done under a standard set of conditions. We have found the GibcoBRL Life Technologies eLONGase PCR reaction kit to work on approximately 80% of our first-pass unoptimized PCR attempts.

We use the eLONGase enzyme mix in a combined master mix format (i.e., Taq polymerase, eLONGase buffers, and dNTPs combined - excluding PCR primers and DNA) and stored frozen at -20° C in a PCR-ready 96-well plate.

After thawing this PCR-ready plate, gene specific forward and reverse primers are added along with target DNA. All primers for first pass PCR are selected using the pcr_overlap program (see DNA Analysis Protocols Manual - PCR Primer Selection and Design).

Equipment

Reagents

Detailed Protocol

I. Single Reaction Setup

Table 1

Table 1.

Reagent	[Initial Conc.]	[Final Conc.]	Volume (µL)
eLONGase Buffer A	10X	0.5X	1
eLONGase Buffer B	10X	0.5X	1
ddH20	10X	0.5X	1
DNTPs	4 mM	100 µM	0.25
eLONGasetaq	1 U/µL	0.4 U/µL	0.4
Primer 1	7 µM	140 nM	0.2
Primer 2	7 µM	140 nM	0.2
DNA	5 µM	2 ng/µL	4
Total Volume			10

II. eLONGase MasterMix Reagent Setup

The eLONGase MasterMix is aliquoted into two columns of a 96-well skirted PCR plate.
A 7-fold volume (39.2 L/well) of MasterMix is placed in columns 1 and 7 in the PCR-ready plate.
We add in one extrea reaction to accommodate pipetting error across the columns.

III. eLongase Master Mix Recipe

For ease of aliquoting to the PCR-ready plate, a large voume MasterMix (12x96 plate volume or greater) can be set up in a deep 96-well plate and transferred using a multi-chanel pipettor (or robot) to the PCR-ready plate (see last column, Table 2, below).

Table 2.

Reagent	[Initial Conc.]	[Final Conc.]	Volume (µL) 1x96 plate (2 columns)	Volume (µL) 12x96 plates (24 columns)
eLONGase Buffer A	5X	0.5X	14	168.0
eLONGase Buffer B	5X	0.5X	14	168.0
ddH20	1X	1X	41.3	495.6
dNTPs	4 µM	100 µM	5.6	42.0
eLONGase Taq	1 U/µL	0.04 µL	0.25	67.2
Total Volume			78.4	940.8

Note: All additions are to a single well in one column of the deep 96-well plate, unless otherwise stated.

IV. Preparation of Elongase MasterMix To Be Aliquoted

Thaw Elongase buffers A and B an ddNTPs. After thawing, vortex well.
Keep Elongase enzyme in freezer until needed.
Add 495.6 L - ddH2O
Add 168 L - Elongase Buffer A
Add 168 L - Elongase Buffer B
Add 42 L - dNTPs
Remove Elongase enzyme from freezer
Add 67.2 L - Elongase enzyme
Slowly mix MasterMix 30 times in each well using a 20-200 L multi-channel pipette (setting=15 L).
Centrifuge deep 96-well block on low speed setting (Jouan Centrifuge: 190 x g for 20 sec, Program 1 (1000 rpm for 20 sec>.

V. Preparation of 96-well Skirted PCR-Ready Plates

Draw a line between columns 6 and 7 to indicate the two halves of the skirted 96-well plate.
Circle the well in row Q and column 1 (in the upper left hand corner) for orientation.
Aliquot 39.w L form each well of the deep 96-well plate to columns 1 and 7 of a PCR-ready 96-well skirted plate using a 20-200 multi-channel pipettor.
Place each finished plate on ice.
Label the aluminum sealing foil with the current date and PCR-READY MASTERMIX.
Seal each plate with aluminum sealing foil and lace in -20 C freezer.

VI. PCR-Ready Amplification Setup

By Hand:

These directions outline the setup of one plate. Two primer sets can be amplified on one plate, one set in columns 1 throug 6 (referred to below as primer set #1) and anothe in columns 7 thorugh 12 (referred to below as primer set #2). Multiple plates can be set up at once, keeping all plates on ice while not in use.

Preheat a Tetrad alpha block.
Thaw one PCR-ready MasterMix plate and the block containing the two sets of primers to be used in the reactions. Centrifuge the primer block on low speed setting (Jouan Centrifuge - 190 x g for 20 sec, Program 1 (1000 rpm for 20 sec).
Using the 8-channel pipettor, transfer 1.4 L of the forward primer of primer set #1 to column 1 of the PCR-ready MasterMix plate.
Using the 8-channel pipettor, transfer 1.4 L of the reverse primer of primer set #1 to column 1 of the PCR-ready MasterMix plate.
Repeat steps 2 an d3 for primer set #2, this time dispensing the primers into column 7 of the PCR-ready MasterMix plate.
Mix the MasterMix and primers in column 1 using the 8-channel pipettor. Change tips and repeat for column 7.
Aliquot the Master Mix using the 8 channel pipettor. Take out 6 L from column one and dispense into column 2. Repeat for columns 3 through 6. Change tips and repeat this process for the Master Mix and primers in column 7. (See Figure 3 below). Centrifuge the plate on low speed setting.
7. Using the Beckman Multimek, add 4 uL of DNA to each reaction.
8. Centrifuge the plate on low speed setting, cover with Microseal A film and place in the MJResearch Tetrad.
Use the folowing cycling conditions:
1. 94° C for 30 sec
2. 94° C for 30 sec
3. 60° C for 30 sec
4. 68° C for 2 min
5. Repeat steps 2-4 34 times
6. 68° C for 5 min

Figure 1. Aliquoting MasterMix and Primers from Columns 1 and 7

A1	2	3	4	5	6	A7	8	9	10	11	12
B1						B7
C1						C7
D1						D7
E1						E7
F1						F7
G1						G7
H1						H7

This figure represents a PCR-Ready Master Mix plate. Columns 1 and 7 contain the Master Mix and Primer mixture.
To aliquot, 6 L will be removed from column 1 and placed in column 2. This step will be repeated for columns 3 through 6.
The whole process will be repeated for columns 7 through 12.

By Robot:

(Refer to PCR 9complex).mpt robot protocol, forthcoming)

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