AlanFung:LabNotes/ASE/2010/2010-7-26

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RNA allelotyping on hybrids[edit]

Exp. #1 Perform CES36k capturing experiment on double stranded cDNAs[edit]

  • Samples:
*Samples:                               
**A: 1000-somatic:  61.9ng/ul x 15ul
**B: 1000-iPS:      87.8ng/ul x 15ul
**C: 1034-iPS:      53.5ng/ul x 15ul
**D: A2:            49.7ng/ul x 15ul
**E: B4:            44.8ng/ul x 15ul
**F: 1016-somatic:  58.1ng/ul x 15ul
**G: 1016-iPS:      60.6ng/ul x 15ul
**H: B5-iPS:        55.5ng/ul x 15ul
**BJ:               27.2ng/ul x 15ul
**HUES6:            40.9ng/ul x 15ul
  • Probes: CES36k18bp, 590nM (prepared by Kun on 7/16/2010)
  • Target:Probe ratio=200. With 200ng cDNA template, we need 1.4ul of probes.

Sample ng/ul Template Probes 10X Buffer H2O
A 61.9 3.23 1.4 1 4.37
B 87.8 2.28 1.4 1 5.32
C 53.5 3.74 1.4 1 3.86
D 49.7 4.02 1.4 1 3.58
E 44.8 4.46 1.4 1 3.14
F 58.1 3.44 1.4 1 4.16
G 60.6 3.3 1.4 1 4.3
H 55.5 3.6 1.4 1 4
BJ 27.2 7.35 1.4 1 0.25
HUES6 40.9 4.89 1.4 1 2.71
  • Add in two drops of mineral oil using P200 pipette
  • Spin down
    Saved under Kun KZ-S-MIP
    95c 30sec -> cool down to 60C at 0.1C/sec -> 60C 20h 
    -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) 
     -> 60C 20h-> 94C 2min -> add 1ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold.
   PCR
   2X iProof Mastermix     50ul      
   AmpF6.3NH2 (100uM)     0.2ul       
   AmpR6.3NH2 (100uM)     0.2ul          
   50X SYBG I             0.4ul      
   H2O                     40ul    
   Captured DNA            10ul
X11
2X iProof Mastermix 550
AmpF6.3NH2 (100uM) 2.2
AmpR6.3NH2 (100uM) 2.2
50X SYBG I 4.4
h2o 440
   98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 20 
   Monitoring the reactions and terminate the program right before the amplification curves reach the plateau.
Purified with Qiaquick columns, quantified with Nanodrop:
   (A) 6.1ng/ul x 40ul
   (B) 9.3ng/ul x 40ul
   (C) 8.4ng/ul x 40ul 
   (D) 7.7ng/ul x 40ul        
   (E) 7.4ng/ul x 40ul  
   (F) 6.0ng/ul x 40ul        
   (G) 4.6ng/ul x 40ul     
   (H) 6.1ng/ul x 40ul
   (Hues6)6.4ng/ul x 40ul
   (BJ)13.5ng/ul x 40ul

File:ZhangLab 2 2010-07-30 12hr 45min.jpg

    • E-gel size Selection for BJ

File:ZhangLab 2 2010-07-30 16hr 03min.jpg

  • Set up 2nd round PCR to add barcodes.
    • Forward primers:
   AmpF6.3Sol:   AATGATACGGCGACCACCGACACTCTCAGATGTTATCGAGGTCCGAC
    • Reverse primers:
   Sample ID       Primer ID     Bardcode (rev. comp.)
   (A)     AmpR6.3Ind1           
   (B)     AmpR6.3Ind2           
   (C)     AmpR6.3Ind3           
   (D)     AmpR6.3Ind4           
   (E)     AmpR6.3Ind5           
   (F)     AmpR6.3Ind6           
   (G)     AmpR6.3Ind7           
   (H)     AmpR6.3Ind8           
   (Hues6) AmpR6.3Ind9
   (BJ)    AmpR6.3Ind10
   PCR                                  x 8
   Template:                       4ul
   2x Kapa SYBG qPCR Master Mix:  50ul
   100uM AmpF6.3Sol:             0.4ul
   100uM AmpR6.3IndXX:           0.4ul
   H2O                            46ul
PCR 1 X11
2X Kapa SYBG qPCR Master Mix 50 550
100uM AmpF6.3Sol 0.4 4.4
H20 46 506
   95C 30sec -> (95C 3sec -> 60C 20sec) x 5 -> 60C 1min
   Purified with Qiaquick columns, quantified with Nanodrop, and pooled in equal ratios.
  Sample
A:     17.4ng/ul x 40ul
B:     28.3ng/ul x 40ul
C:     24.7ng/ul x 40ul
D:     22.1ng/ul x 40ul
E:     21.2ng/ul x 40ul
F:     20.2ng/ul x 40ul
G:     16.7ng/ul x 40ul
H:     16.9ng/ul x 40ul
Hues6: 20.7ng/ul x 40ul
BJ:    17.9ng/ul x 40ul
ng/ul To get 100ng (ul)
A 17.4 5.75
B 28.3 3.53
C 24.7 4.05
D 22.1 4.52
E 21.1 4.74
F 20.2 4.95
G 16.7 5.99
H 16.9 5.92
Hues6 20.7 4.83
BJ 17.9 5.59
Total Volume 49.87
Concentration 20ng/ul
    • Size Selection