Rui:LabNotes/SingleCell/2011-1-24: Difference between revisions

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=PCR based amplification=
PCR based amplification


==1.prepare samples==
==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
  *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 33: 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]

[1]

[2]

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

PCR result check[edit]

[3]