Glutathione S-transferase Primer Pair for RT-PCR (reverse transcription followed by polymerase chain reaction) analysis of mRNA expression.
Primer Pairs for RT-PCR provide convenient primers for analyzing the expression of specific mRNAs or got specific PCR fragment by RT-PCR. Each Primer Pair includes a 5´ and 3´ oligonucleotide primer provided in individual vials at 50¦ÌM ready for addition to the amplification reaction. The Primer Pairs have been confirmed in two-step conventional RT-PCR using Cloning vector pGEX-6P-1.
PCR products from the use of the Primer Pairs have been sequence-confirmed for appropriate.
• Thaw all reagents completely on ice before use.
• Use either random primers or oligo (dT) for reverse transcription.
• All PCR reactions should be assembled on ice.
• The recommended annealing temperature is 52¡ã C.
• To minimize the risk of amplicon contamination of the Primer Pair and other PCR reagents, the following is recommended:
PCR reactions should be set up in an area separate from where PCR products are analyzed.
Pipettes and tube racks should be specifically designated for PCR.
Use aerosol barrier pipette tips.
• Follow the steps below to determine the number of PCR reactions and to calculate the amount of PCR master mix necessary.
1. Determine the number of cDNA samples that will be analyzed.
2. Add 2 to the number of cDNA samples to account for a negative and a positive control.
3. Multiply the number determined in step 2 by 1.1 to account for pipeting error. This is the number of reactions for which the master mix should be made.
For example, the master mix for 3 cDNA samples should be:
1. 3 cDNA samples will be analyzed
2. Plus 2 for negative and positive controls = 5
3. 5 x 1.1 for pipeting error = 5.5
• Primer Pairs are not validated for use in kinetic RT-PCR.
• Dilute RT reaction five-fold before using in the PCR reaction.
The Primer Pair is stable for up to one year at
-20¡ã C in a non-frost free freezer.
Avoid repeated freeze-thaw cycles.