Real-time quantitative PCR experiment (Comparison of gene expression)

Transcription

1 Real-time quantitative PCR experiment (Comparison of gene expression) 0 PRECAUSION Use filter tips for pipetting. Use longer tip if possible (e.g. use P20 tip for taking 5 L, not P2-short tip.) Be mindful to avoid any contact of pipette nose with tube rim. Dispose a pipette tip when you treat any thick DNA solution with it. We compare sample A (control) and B (drug-treated). (Sample C and D are positive controls.) 1 SET UP Each group has charge of or. Label all 1.5mL tubes as below. Take 1mL of water in the tube labelled water. Water STD1 DNA 1 Water STD1 1:100 STD2 DNA 2 1:100 STD2 1:1000 STD3 DNA 3 1:1000 STD3 Premix STD4 DNA 4 Premix STD4 A STD5 DNA 5 A STD5 NC NC B B C C D D Each group will have charge one of them ( or ). DNA 1 DNA 2 DNA 3 DNA 4 DNA

2 In this final experiment, you have to choose the range of the standard calibration curve according to the result of 2 nd PCR experiment. For today, we will use STD1 to STD5 from the 2 nd experiment. Take 198 L of water to the tube labelled *** 1:100. Take 90 L of water to the tube labelled *** 1:1000. Take 45 L of water in tubes labelled *** DNA1 through *** DNA5. 2 MAKING PREMIX Add following reagents in the tube labelled ~premix. Make slightly excess volume (equivalent to one PCR reaction) than required. ddh 2 O 200 2x Thunderbird Master Mix x ROX M Primer mix. ( or ) 15 ( L) Total 475 Vortex and spin down. Aliquot 38 L each in the tubes labeled xxx STD and xxx NC. Aliquot 57 L each in the tube labeled A (B, C, D) or A (B, C, D). Add 2 L of water in the xxx NC tube, vortex and spin down. Keep the tubes with premix in a drawer

3 3 MAKING DNA SOLUTION SERIES FOR STANDARD CURVE. (The followings are already done in the previous step) Take 198 L of water to the tube labelled *** 1:100. Take 90 L of water to the tube labelled *** 1:1000. Take 45 L of water in tubes labelled as xxx DNA1 through xxx DNA5. The PCR product from Pre-Test 1 will be the standard DNA concentrate. For this time, use the PCR product of sample A. (IMPORTANT)Bring the standard DNA concentrate at this point for first time to avoid any contamination to happen. Make standard DNA solution by diluting the standard DNA concentrate. Take 2 L of the standard DNA concentrate in the 1:100 tube, vortex and spin down. Take 10 L of the 1:100 solution in the 1:1000 tube, vortex and spin down. Wash your gloves or change them to avoid possible contamination. Add 5 L of DNA1 solution to DNA2 tube (containing 45 L of water). Use P20 tip to take the 5 L. With P2-short tip, the pipette nose would contact with tube rim potentially contaminated with concentrated DNA. Mix by vortexing. Spin down if the solution is attached on the behind the lid. Add 5 L of DNA2 to DNA3 tube, vortex and spin down in the same manner. Repeat the same thing trough DNA

4 4 MAKING REACTION MIXTURE Add 3 L of sample A cdna (the ones reverse transcribed yesterday) in the tube labelled A or A, which containing the premix to make the reaction mix solution for sample A. Mix by vortexing and spin down. Do the same thing for sample B, C, and D (add 3 L to the labelled tube, mix and spin down) to make the reaction mix solution for sample B-D. For standard DNA reaction mix, start from DNA5, which has least DNA content. Add 2 L of DNA5 solution to STD5 tube. Mix by vortexing and spin down. Do the same thing for STD4 through STD1. Wash your hands with gloves on or change gloves. 5 ALIQUOT IN PCR TUBES (See Supplementary Table 2 for aliquot pattern.) Set four 6-well PCR strips on a tube rack. Set them vertically as before. NC reaction mix will be face the far end and the STD1 in near end. Make a small label to distinguish which is which if necessary. Also, make a label to distinguish Samples A to D. Aliquot the mixture containing cdna of Sample A, B, C, and D, respectively, into three tubes (20 L each) to make triplicates

5 Apply 20 L each of NC reaction mix in the far end of two 6-well strips. Apply 20 L each of STD5 reaction mix in the second far of the 6-well strips. When making aliquots, try not to move around your pipette above the alreadyaliquoted wells having thinner DNA in order to minimize any possible contamination form your tip that might contain thicker DNA. This is the frequent cause of contamination in NC and thin DNA reaction mix. Aliquot STD4 through STD1 in the same way (20 L each). Close the tubes with lids. Spin them down and apply to the real-time PCR system (StepOne Plus). Start the reaction. (If you don t start the reaction immediately, keep them in dark and at 4 in a refrigerator.)