Rui Liu

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Lab Projects

Sample record for sequencing

6.8.11: RL-Hap_RNAseq-1_13-Jun6

Sample ID IndX
Hap1 Nextera ID1
Hap2 Nextera ID2
Hap3 Nextera ID3
Hap4 Nextera ID4
Hap5 Nextera ID5
Hap6 Nextera ID6
Hap7 Nextera ID7
Hap8 Nextera ID8
Hap9 Nextera ID9
Hap10 Nextera ID10
Hap11 Nextera ID11
Hap12 Nextera ID12
RNA-ER PCR_R.N2Ind12
RNA-EL PCR_R.N2Ind13


Hap samples (1-12) prepared on lab note 6.6.11 [1]. Briefly, Twelve cell lysate aliquots (~1pg gDNA) were amplified with Nextera adapter mix (barcode 1-12) and mixed the 12 samples at 1:1 ratio.
RNA samples prepared on lab note [2]. Briefly, ES (~50cells) samples from Dr. Yi Zhang lab were purified with RNA by Zymo kit (named ER sample) or directly lysated (named EL sample), and then amplified with PCR_based protocol, which introduces poly(dT) to 5' end and poly(dA) to 3' end of the ds cDNA. Solexa Y adaptor was ligated to the ends and illumina barcoded primers (N2.ID12 and N2.ID13) were used to amplfied ER and EL samples, respectively.
Finally, Hap samples and RNA samples were mixed as 10:1:1 ratio for sequencing.

Manuals

Sequencing for Genewiz

Prepare for sequencing (Genewiz): 20ng DNA in 10ul total volume + 5ul 5uM primer

Zeroblunt cloning

TA cloning

Vector map [3]

Manual File:Original TA cloning kit.pdf

Bio-Rad QPCR machine

  1. Program on desktop: "Opticon Moniter 3"
  2. Instrument: Chromo4:CD003042. "Instrument"-->"Scan for instruments" or "Quick load"(under "Master")-->choose "Chromo4:CD003042"
  3. "Prepare new run" or "open" a old file and "edit"
  4. "Plate setup"-->"edit"-->select "samples" and "SBG1" for SYBR green I
  5. "Protocol setup"-->"edit"-->add "plate read" before "repeat cycle"
  6. "Melting curve" optional, hold at 15C, volume 20ul
  7. "Run" and "Save", files are saved in "Opticon Users 3"/Rui
  8. "Status" and "Quantitation" are used for monitoring PCR process
  9. "Quantitation"-->"copy to clipboard"-->"Data graph" and edit under "Paint"

Ep Realplex QPCR machine

  1. "Mastercycler ep realplex" on desktop
  2. log in User name: "EPPENDORF", password: "e"
  3. "PCR program"-->direct change temp and time-->right click to insert and advance edit
  4. "Plate layout"-->"Filer 520nm"-->"SYBR"; "Sample volume", "Probe"-->"SYBR green", "Background"-->"Biorad strip tube-50ul" (Attention!); select sample area--> right click-->"unknown"-->ID
  5. "Save as" a "template" or an "assay"
  6. "Set up"-->"System Configuration"-->"Cycler"-->"Lid"-->select "Heat"-->Okey and close for preheat the lid
  7. "Monitoring" for PCR process
  8. "Analysis data" and save as assay

Microscope