Difference between revisions of "Real Time PCR From Cell Culture"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (→References (Saltiel Lab): added Chang et al Mol Endocrinol) |
|||
| Line 11: | Line 11: | ||
==Protocol== | ==Protocol== | ||
===RNA Extraction=== | ===RNA Extraction=== | ||
| − | #Use RNEasy kit with Qiashredder. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer). | + | #Use RNEasy kit with Qiashredder. see [[Harvesting RNA from Cells grown in monolayer]]. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer). |
#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen. | #Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen. | ||
#Store at -20 until RT reaction | #Store at -20 until RT reaction | ||
Revision as of 15:25, 12 May 2009
Contents
Real Time qPCR
Materials
- cDNA for templates
- Qiashredder and RNEasy kits from Qiagen
- Superscript Kit from Invitrogen
- SyberGreen PCR Master Mix Applied Biosystems
- 96 well qPCR plate
- Primers (Dilute to 0.22 uM mixture of fwd and rev)
- Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html
Protocol
RNA Extraction
- Use RNEasy kit with Qiashredder. see Harvesting RNA from Cells grown in monolayer. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
- Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
- Store at -20 until RT reaction
RT-PCR Reaction
- Use 8 uL of RNA per RT reaction.
- Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
- Store cDNA at -20 until use
Plate Preparation
- Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine
- Get 96 well block and keep on rack. Do not touch bottom of plate.
- Add 1 uL template per well.
- Add 9 uL primer per well
- Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well
References (Saltiel Lab)
<pubmed>18829989</pubmed> <pubmed>17008399</pubmed> <pubmed>17200717</pubmed> <pubmed>17192460</pubmed> <pubmed>16926380</pubmed>
