Rui:LabNotes/SingleCell/2011-1-24: Difference between revisions
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PCR based amplification | PCR based amplification | ||
==RNA samples and PCR protocol== | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2011-1-20] | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:SingelCell#Protocols] | |||
==Prepare samples== | |||
*Follow cell lysis buffer w/o NP40 | |||
*8 samples, 10 reaction mixes (esp. for small amount) | |||
*65C, 90sec | |||
*Sample arrangement in 8 tube strip: Hues6-ES,blank,blank,blank,Hues6-EB, blank,blank,blank | |||
*Sample concentration: NTC (0.5ul Nf water), 10pg (0.5ul #5), 100pg (0.5ul #4), 500pg (0.5ul #3); one strip one concentration | |||
==1st cDNA== | |||
==Primer removal== | |||
*Exonuclease I add 0.025ul (20U/ul) per reaction, adjust Nf-water | |||
==PolyA tailing== | |||
*Original tube is divided into additional 3 tubes | |||
*Sample arrangement in 8 tube strip: Hues6-ES,-ES,-ES,-ES,Hues6-EB, -EB,-EB,-EB | |||
==2nd cDNA== | |||
*Prepare 9 reactions should be enough | |||
==PCR== | |||
*Dilute SYBR green I to 20X, then add 0.2X to PCR tube | |||
*Run PCR tube in BioRad DNAEngine by using Opticon Monitor 3 program | |||
*If no automatic connection, click on "Instrument" --> "Scan for instrument" | |||
*If no "prepare new run" under "Master", click on "open" --> open an old file, then edit | |||
*When editing the plate setup, choose SBG1 for SYBR green I detector | |||
*When editing protocol setup, add "plate read" before repeating the cycle | |||
*When hit run, it automatically asks for saving the running file | |||
*After a few running cycles, check "Status" and "Quantitation" to visualize PCR process | |||
*20 cycles need more than 3hrs to finish | |||
*No saturation was observed in any samples after 20 cycles, even for 500pg | |||
*NTC showed similar amplification as other samples, with a broader range of curves | |||
==PCR result check== | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/SingleCell/2011-1-25] |
Latest revision as of 19:14, 24 February 2011
PCR based amplification
RNA samples and PCR protocol[edit]
Prepare samples[edit]
*Follow cell lysis buffer w/o NP40 *8 samples, 10 reaction mixes (esp. for small amount) *65C, 90sec *Sample arrangement in 8 tube strip: Hues6-ES,blank,blank,blank,Hues6-EB, blank,blank,blank *Sample concentration: NTC (0.5ul Nf water), 10pg (0.5ul #5), 100pg (0.5ul #4), 500pg (0.5ul #3); one strip one concentration
1st cDNA[edit]
Primer removal[edit]
*Exonuclease I add 0.025ul (20U/ul) per reaction, adjust Nf-water
PolyA tailing[edit]
*Original tube is divided into additional 3 tubes *Sample arrangement in 8 tube strip: Hues6-ES,-ES,-ES,-ES,Hues6-EB, -EB,-EB,-EB
2nd cDNA[edit]
*Prepare 9 reactions should be enough
PCR[edit]
*Dilute SYBR green I to 20X, then add 0.2X to PCR tube *Run PCR tube in BioRad DNAEngine by using Opticon Monitor 3 program *If no automatic connection, click on "Instrument" --> "Scan for instrument" *If no "prepare new run" under "Master", click on "open" --> open an old file, then edit *When editing the plate setup, choose SBG1 for SYBR green I detector *When editing protocol setup, add "plate read" before repeating the cycle *When hit run, it automatically asks for saving the running file *After a few running cycles, check "Status" and "Quantitation" to visualize PCR process *20 cycles need more than 3hrs to finish
*No saturation was observed in any samples after 20 cycles, even for 500pg *NTC showed similar amplification as other samples, with a broader range of curves