Noi/NOTES/2011-10-6

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Revision as of 17:55, 8 October 2011 by >Noi (→‎Probe calculation)
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Plan of probe (from LC Bioscience) production

  • Follow the protocol: [1] and standard protocol using to make DMR330k probe set
  • After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
  • perform expansion PCR to amplify the oligoes as the template
  • 2011_10_07 received oligo mix from LC Science
  • Oligo info.: DNA oligo mix order 4617, concentration 14.94ng/ul-->490.65nM (ssDNA, size 100nt), volume 25ul
  • Dilute to 20nM volume 100ul by mixing 4.08ul of oligo with 95.92ul H2O

Expansion PCR

Components Volume (ul) Final conc.
20nM LC Sciences Oligoes 10.00 1nM
eMIP_CA1_F (100uM) 0.80 400nM
eMIP_CA1_F (100uM) 0.80 400nM
2x Kapa SYBG MM 100.00 1x
H2O 88.40
Total 200.00

Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold

  • Primer info.
    • eMIP_CA1_F: TGCCTAGGACCGGATCAACT (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C
    • eMIP_CA1_R: GAGCTTCGGTTCACGCAATG (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C
  • Note:
    • The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer
    • The number of cycles will be monitored

File:Expansion-PCR 2011 10 07.png

  • Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  • Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM
  • Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification
  • Perform production PCR

Production PCR

Components 1 rxn 32x rxn mix
1st round amplicon (10nM) 0.20 6.40
eMIP_CA1_F (100uM) 0.40 12.80
eMIP_CA1_R (100uM) 0.40 12.80
2x Kapa SYBG MM 50.00 1600.00
H2O 49.00 1568.00
Total 100.00 3200.00

Program
95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 20sec) x 13 -> 72C 2min -> 15C hold

  • The number of cycles will be monitored to prevent overamplification

File:ProductionPCR-LC 2011 10 07.png

  • Dr. Zhang suggested to check the oligoes by running 2ul oligoes before amplification in 6% TBU gel and quantify concentration if it correspond to the concentration specified on the tube. Also run the 1st round amplicon (after column purification) and amplified amplicon (before purification)

File:LC-PQ 2011 10 07.png

  • A1 = single strand LC oligoes before amplification 1.8ul
  • B1 = 1st round amplicon after column purification 1.8ul (total volume 100ul)
  • C1 = amplified amplicon before EtOH precipitation & column purification 1.8ul
  • Note: A2, B2 & C2 = load sample 0.2ul to prevent intensity saturation when do quantification (haven't done yet)
  • From the gel image, the concentration of oligoes from LC Sciences may be overestimated since it showed a very faint band at the 100 bp band (the expected amount of 1.8ul ~ 25ng), and I also observed the smear band along the lane. For the 1st round amplicon and amplified amplicon, they showed the specific band ~110bp as expected.
  • Perform EtOH precipitation of the 32 rxn PCR in 4x 15mL tube
    • 800ul amplified amplicon +80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH
    • incubate at -80C for 30min --> centrifuge at 3,000 rpm for 30 min, wash with 75% EtOH
    • resuspend with 100ul each tube
    • re-purify with Qiaquick column (4 columns) and elute with 50ul EB buffer each --> total volume 200ul purified amplicon
    • measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ng. The yield seems low when compare to the DMR330k preparation (ave ~30ug for 32 rxn, started with the same amount of template), but in this batch I stopped the reaction earlier (13cycles) and the size of product is smaller.

Nicking exonuclease digestion

  • Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4 tube reaction)
  • Heat inactivate enzyme at 80C for 20 min
  • Add 10ul Nb.BrsDI (10U/ul) at 65C for 1 hr
  • Purify with 6 Qiaquick colums, elute with 50ul EB buffer each --> total volume 300ul (Note: use more Qiaquick columns since the volume of of reaction was large, total 800ul and doing multiple pass of DNA through the column didn't help to recover DNA very well)
  • Measure the conc. of nicked DNA with Nanodrop -->50.4ng/ul, yields = 15.1ug (why recovery rate so high > 85%?)
  • Before doing PAGE size selection to purify the 70nt band, I ran the gel to check if DNA nicking work well by loading the same amount of DNA before and after incubation with each enzyme at the same amount

File:LC-probe-Nicking 2011 10 07.png

  • A = amplified amplicon before nicking exonuclease digestion
  • B = After incubation with Nt.AlwI
  • C = After incubation with Nb. BsrDI
  • Note: The digestion was not very complete since I still see the partial digested band of ~ 90nt. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high and I try to minimize the volume of reaction. However in this experiment I have no time to optimize and I think that the expected band (~70nt or slightly lower) was a lot brighter than the incomplete digested band, so I continued to do PAGE size-selection to purify the 70nt band in 6% TBU gel.
  • PAGE quantification will be performed to check the efficiency of each enzyme by comparing the ratio of the intensity of each band.

File:ZhangLab 2 2011-10-08 00hr 11min PAGE-SS-1.jpgFile:ZhangLab 2 2011-10-08 00hr 15min PAGE-SS-2.jpg

  • I used the old TBU gel prepared 2 weeks and almost 1 month before, the resolution of the DNA was not very good. The gel prepare 2 week gave the a better resolution the 1 month prepared gel. Would recommend to use the fresh prepared gel or not > 2 weeks if there is no time restriction.
  • I incubated the gel eluted DNA overnight with EtOH, NaOAc, and Glycoblue at -80 --> will precipitate and quantify concentration on Saturday 08, 2011

Plan for Saturday

Capture set up

  • Perform bisulfite conversion of PGP1F, PGP1iPS, and H1 to be enough for 4 capture reactions each (to optimize annealing temperature)
  • Bis-cvt DNA ~ 300ng/rxn if it's enough (may use 250ng if the total yield is not enough)
  • Probe/ target ratio: 1,000/1 (since the probe size is very small (4,000), so to increase capture efficiency, Dr. Zhang suggested to increase the probe to target ratio.

Probe calculation

Probe/target 1000:1 '
Probe size 4,000
Template 300.00 ng
Human gDNA MW 1.820E+12 g/mole
Human gDNA (300ng) 1.648E-19 mole
Probe required 1.648E-16 mole
Probe MW 8.649E+07 g/mole (4,000x70bpx308.9D/bp+79D )
Amount probe required 1.426E-08 g
Amount probe required 14.26 ng


  • Expected probe amount required for ~12-15 rxn = 214ng

Capture plan

  • Capture volume --> in 15-20ul
  • Vary annealing temperature --> try all below 55C we are currently using for DMR330k BSPP capture --> 47.5C, 50.0C, 52.5C, 55.0C ??? please suggest

Program
95c 30sec -> cool down to X C at 0.02C/sec -> X C 20h

  -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) 
  -> X C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.