Rui:LabNotes/SingleCell/2011-1-24: Difference between revisions
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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: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 | *Follow cell lysis buffer w/o NP40 | ||
*8 samples, 10 reaction mixes (esp. for small amount) | *8 samples, 10 reaction mixes (esp. for small amount) | ||
Line 36: | Line 38: | ||
*No saturation was observed in any samples after 20 cycles, even for 500pg | *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 | *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