Basic pcr

EXPERIMENTAL OBJECTIVES

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented PCR Protocol.

methodology

MyTaq qPCR
Primers were designed and obtained using the Oligosynthesis Quick Instruction.  Primers annealed to complementary sequences in the pUC19 plasmid (New England Biolabs) with a predicted amplicon size of 1,010 bp.  PCR was performed using 5 µl of template DNA (1 pg/µl ), 2.5 µl forward primer, 2.5 µl reverse primer (both 10 µM).  Total reaction volume was 50 µl made up using 25µl of 2x MyTaq Mix (Bioline) and 15 µl of distilled water.  Thermocycling conditions were 95 °C, 2 minutes; [98 °C,10 seconds; 50 °C, 20 seconds; 72 °C, 20 seconds] x30 with a final 5 minute incubation at 72 °C. 

Phusion PCR
Primers were designed and obtained using the Oligosynthesis Quick Instruction.  Primers annealed to complementary sequences in the pBABE-puro plasmid [1]   generating a predicted amplicon size of 2895 bp.  PCR was performed using 3.125 µl of template DNA (10 ng/µl ), 2.5 µl forward primer, 2.5 µl reverse primer (both 10 µM), 10 µl x10 High-Fidelity buffer, 1 µl dNTPs (10 mM each), 0.5 µl Phusion polymerase (buffer, nucleotides andenzyme all New England Biolabs) and made up to a final volume of 50 µl with distilled water.  Thermocycling conditions were 95 °C, 30 seconds; [98 °C,10 seconds; 68 °C, 20 seconds; 72 °C, 90 seconds] x30 with a final 5 minute incubation at 72 °C.

results

MyTaq PCR was employed for successful short-range (up to ~1 Kbp) reactions.  Phusion PCR successfully generated amplicons of multiple kilobases.

Figure 1  MyTaq PCR and corresponding negative control.  MyTaq PCR executed using the PCR Protocol.  Twelve microliters of total reaction volume were analyzed by gel electrophoresis as part of the PCR Protocol for identification of target amplification.  Expected amplicon size: 1010 bp.

Figure 1  MyTaq PCR and corresponding negative control.  MyTaq PCR executed using the PCR Protocol.  Twelve microliters of total reaction volume were analyzed by gel electrophoresis as part of the PCR Protocol for identification of target amplification.  Expected amplicon size: 1010 bp.

Figure 2   Phusion PCR and corresponding negative control.  Phusion PCR executed using the PCR Protocol. Ten microliters of total reaction volume were analyzed by gel electrophoresis as part of the PCR Protocol for identification of target amplification.  Expected amplicon size: 2895 bp. 

Figure 2   Phusion PCR and corresponding negative control.  Phusion PCR executed using the PCR Protocol. Ten microliters of total reaction volume were analyzed by gel electrophoresis as part of the PCR Protocol for identification of target amplification.  Expected amplicon size: 2895 bp. 

 

[1]  Morgenstern JP, Land H. Advanced mammalian gene transfer: high titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990;12:3587-96


VALIDATION DATE:  August 2015