Noi/NOTES/2011-10-6: Difference between revisions

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== Capture plan ==
== Capture plan ==
* Capture volume --> in 15-20ul  
* 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
* Vary annealing temperature --> try all below 55C we are currently using for DMR330k BSPP capture (since the probe size is shorter) --> 47.5C, 50.0C, 52.5C, 55.0C ??? please suggest
'''Program'''<br>
'''Program'''<br>
95c 30sec -> cool down to X C at 0.02C/sec -> X C 20h  
95c 30sec -> cool down to X C at 0.02C/sec -> X C 20h<br>
   -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)  
   -> 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.
   -> X C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
* Note: I try not to change the capture condition this time since want to make sure that everything will work as in the past except varying the annealing temp.

Revision as of 17:58, 8 October 2011

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 (since the probe size is shorter) --> 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.
  • Note: I try not to change the capture condition this time since want to make sure that everything will work as in the past except varying the annealing temp.