Polymerase Chain Reaction Protocol
This is a standard PCR protocol used on all first pass (unoptimized) PCR amplifications. This protocol outlines:
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.
- Setup of a single PCR reaction
- Preparation of PCR-ready 96-well plates with Elongase MasterMix
- PCR setup for amplification
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).
DNA Engine Tetrad (MJ Research PTC-225)
Jouan Centrifuge (CR422)
Skirted 96-well plates (MJResearch MSP-9601)
Deep 96-well plate (Eppendorf 0030127 544)
Microseal A Film (MJResearch MSA-5001)
8 channel pipettor (Eppendorf)
Multimek 96 Automated 96-Channel Pipettor (Beckman Instruments)
eLONGase Enzyme Mix (GibcoBRL Life Technologies Cat. No. 10480-028)
dNTPs (Roche Molecular Biochemicals, 1051466, 1051458, 1051440, 1051482) - 4mM
Forward/Reverse oligonucleotide primers - 7µM = 100 ng/µL of 40-mer primer with 5' tailed
universal extension (see DNA Analysis Protocols Manual - PCR Primer Selection/Design) (GibcoBRL Life Technologies)
I. Single Reaction Setup
The total volume for a single PCR reaction is 10 µL. Table 1 lists the reagents
for one reaction and the initial and final concentrations.
Table 1. Reagent Concentrations and Volumes for a Single PCR Reaction
|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.
Note: The reagent setup is specific to our PGA DNA Panel (see DNA Panel) block setup, which consists of a 96 format deep well block containing 48 DNA sample duplicted on either side of the block, columns 1-6 and 7-12, respectively (see Figure 1).
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.eLONGase MasterMix Concentration and Volumes
|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
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:
- 94° C for 30 sec
- 94° C for 30 sec
- 60° C for 30 sec
- 68° C for 2 min
- Repeat steps 2-4 34 times
- 68° C for 5 min
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.
(Refer to PCR 9complex).mpt robot protocol, forthcoming)
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