AlanFung:LabNotes/ASE/2010/2010-8-16

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RNA allelotyping on hybrids[edit]

Exp. #3 Perform CES36k capturing experiment on ds-cDNAs[edit]

ds cDNA synthesis[edit]

*Samples:                                  Nanodrop
    • H1:Hybrid 1 p26: 1800ng/ul x 30ul 1393ng/ul
    • 1034-somatic: Egg donor somatic: 100ng/ul x 130ul 100.1ng/ul

gdna[edit]

    • H1: 240ng/ul x 100ul 175.5ng/ul
   Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
H1 1034s
RNA 3 43
10X Dnase I Buffer 5 5
Rnase-Free Dnase I 2 2
H2O 40 0
   37C 10min 
   Add 4 volumes of RNA binding buffer;
   Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s;
   Add 200ul RNA Wash buffer, spin @15k rpm for 30s;
   Add 10ul DNase/RNase-free water, spin @15k rpm for 30s;
   Yield:
   H1: 310.6ng/ul x 10ul
   1034s: 336.4ng/ul x 10ul 
   Reverse transcription 
H1 1034s
RNA 8 8
10mM dNTP 1 1
50uM dT12-18 1 1
  65C 5min -> chill on ice for 1min
  Add  
	        H1	1034s
10X RT buffer	2	2
25mM MgC12	4	4
0.1M DTT	2	2
RnaseOUT	1	1
Superscript III	1	1
Incubate all tube at 50C for 50min.
  2nd strand synthesis saved under Alan>2ss
  Add 
  5X 2nd strand buffer:  30ul         
  10mM dNTP               3ul       
  DNA Pol I(10U/ul)       4ul          
  RNase H(2U/ul)          1ul         
  H2O                    92ul       
  16C 2h -> 70C 10min -> add 2ul RNase I -> 37C 30min 
  Purified with MinElute columns.
   Yield:
   H1: 252.5ng/ul x 15ul
   1034s: 285.8ng/ul x 15ul

Sample volume TBE Buffer 6X Loading Dye Total Volume /4

H1+gDNA	14.73	        16	        28	        58.73	        14.68
1034+cDNA	27.75	        4	        28	        59.75	        14.94

RNA allelotyping on hybrids[edit]

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

  • 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
H1 (cDNA)	252.5	0.79	 1.4	1	    6.81
1034s (cDNA)	285.8	0.7	 1.4	1	    6.9
H1 (gDNA)	175.5	1.14	 1.4	1	    6.46


  • 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.
  • Perform one step qPCR using Indexing seq. primer
PCR	
Captured DNA 1034s (cDNA)	10
2X Kapa SYBG qPCR Master Mix	50
100uM AmpF6.3Sol	        0.4
100uM AmpR6.3Ind11	        0.4
H2O	                        46
   
   98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
   Monitoring the reactions and terminate the program right before the amplification curves reach the plateau.
  • Stop at cycle 12

File:ZhangLab 2 2010-08-19 12hr 33min.jpg

  • One step PCR worked well, move on with the 2 remaining samples
  • Perform one step qPCR using Indexing seq. primer
PCR	
Captured DNA 1034s (cDNA)	10
2X Kapa SYBG qPCR Master Mix	50
100uM AmpF6.3Sol	        0.4
100uM AmpR6.3Ind*	        0.4
H2O	                        46
   
   98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40
   Monitoring the reactions and terminate the program right before the amplification curves reach the plateau.
AmpR6.3Ind1  - H1 gDNA
AmpR6.3Ind12 - H1 cDNA
  • TBU Gel Quantification
Pre-run TBU gel

75C 7 min -> cooling rack 5 min -> Load gel

File:ZhangLab 2 2010-08-19 17hr 19min.jpg


Purified with Qiaquick columns, quantified with Nanodrop and pooled in equal ratios.
   H1(cDNA) 21.3ng/ul x 30ul
   H1(gDNA) 26.9ng/ul x 30ul
   1034s    32.0ng/ul x 30ul
Pool H1(gDNA) and H1(cDNA) in equal ratios
	ng/ul	To get 100ng
H1cDNA	21.3	4.69
H1gDNA	26.9	3.72
Pool H1 (gDNA/cDNA mix) with gDNA mix in 4:6 ratio
Pool 1034s (cDNA) with cDNA mix in 2:8 ratio

	        ng/ul	Ng Needed	Volume
H1 mix	        23.78	60	        2.52
1034s	        32	30	        0.94
cDNA Mix	20	120	        6
gDNA Mix	77.7	90	        1.16


    • Size Selection
Sample volume TBE Buffer 6X Loading Dye Total Volume /4
H1+gDNA 14.73 16 28 58.73 14.68
1034+cDNA 27.75 4 28 59.75 14.94

Turn on centrifuge let it cool down to 4C

  • Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
  • Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
  • Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
  • Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
  • Let dry in hood ~ 10 min.
  • Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.

Store at 4C if needed, spin down before proceeding

  • Qubit Quantification
cDNA-2.42ng/ul (22.1nM)
gDNA-1.19ng/ul (10.87nM)