Difference between revisions of "Real Time PCR From Cell Culture"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with '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 plat...') |
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| − | Real Time qPCR | + | ==Real Time qPCR== |
| − | Materials | + | ===Materials=== |
| − | cDNA for templates | + | *cDNA for templates |
| − | Qiashredder and RNEasy kits from Qiagen | + | *Qiashredder and RNEasy kits from Qiagen |
| − | Superscript Kit from Invitrogen | + | *Superscript Kit from Invitrogen |
| − | SyberGreen PCR Master Mix Applied Biosystems | + | *SyberGreen PCR Master Mix Applied Biosystems |
| − | 96 well qPCR plate | + | *96 well qPCR plate |
| − | Primers (Dilute to 0.22 uM mixture of fwd and rev) | + | *Primers (Dilute to 0.22 uM mixture of fwd and rev) |
| − | Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html | + | *Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html |
| − | 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). | |
| − | + | #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)== | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | References (Saltiel Lab) | + | |
<pubmed>18829989</pubmed> | <pubmed>18829989</pubmed> | ||
Revision as of 19:41, 1 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. 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>
