Real Time PCR From Cell Culture: Difference between revisions

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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===
1.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.  For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
2.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.
3.Store at -20 until RT reaction
#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


RT-PCR Reaction
===Plate Preparation===
1.Use 8 uL of RNA per RT reaction.
#Prepare dilutions of primers. Need 9 uL per well.  Book 3h on qPCR machine
2.Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
#Get 96 well block and keep on rack.  Do not touch bottom of plate.
3.Store cDNA at -20 until use
#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


Plate Preparation
==References (Saltiel Lab)==
1.Prepare dilutions of primers.  Need 9 uL per well.  Book 3h on qPCR machine
2.Get 96 well block and keep on rack.  Do not touch bottom of plate.
3.Add 1 uL template per well. 
4.Add 9 uL primer per well
5.Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well
 
References (Saltiel Lab)
<pubmed>18829989</pubmed>
<pubmed>18829989</pubmed>

Revision as of 19:41, 1 May 2009

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

  1. 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).
  2. Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
  3. Store at -20 until RT reaction

RT-PCR Reaction

  1. Use 8 uL of RNA per RT reaction.
  2. Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
  3. Store cDNA at -20 until use

Plate Preparation

  1. Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine
  2. Get 96 well block and keep on rack. Do not touch bottom of plate.
  3. Add 1 uL template per well.
  4. Add 9 uL primer per well
  5. Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well

References (Saltiel Lab)

<pubmed>18829989</pubmed>

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