Noi/NOTES/2011-10-6

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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 endonuclease digestion

  • Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions)
  • 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 endonuclease 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
  • After EtOH precipatition and washing, resuspend the probe with 15ul each tube --> total volume 150ul
  • PAGE quantification in 6% TBU gel (mix 2ul of probe in total 80 u and load different volume to estimate DNA concemtration)

File:LC-probe-PQ-1.png

  • Final conc. = 4.10ng/ul --> total tields = 150ulx4.1ng/ul = 615ng (lower than expected)

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). Re-measure gDNA conc. before bisulfite conversion.
  • 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), 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) --> 48C, 50, 52C, 55C

Program
95c 30sec -> cool down to 48-54 C at 0.02C/sec -> X C 17.5h
-> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 48-54 C 17h-> 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 well as in the past except varying the annealing temp.

Amplification

  • To test whether the capture work and what is the best annealing temperature for this probe set, I ran the PCR in small volume 25ul and using 2ul of circularized DNA template.
Components 1 rxn 14rxn mix
Captured DNA template 2.00 0.00
10uM CP-2-FA 0.50 7.00
10uM CP-2-RA 0.50 7.00
2xKAPA MM 12.50 175.00
H2O 9.50 133.00
Total 25.00 350.00
  • Aliquot 23ul, add 2ul of DNA template

Program
98C 30sec--> (98C 10sec --> 52C 30sec --> 72C 30sec)x5 cycles --> (98C 10sec --> 60C 30sec --> 72C 30sec)x15 cycles--> 72C 3 min --> 15C hold
File:2011 10 10-test capture20cycles.png

Samples Ct 48C Ct 50C
PGP1F 16.77 16.92
PGP1iPS 16.72 17.12
H1 16.51 16.81


  • To verify the PCR product, 2 ul of each samples were loaded in 6% TBE gel

File:ZhangLab 2 2011-10-10 17hr 30min Amp-CP-2.jpg

  • The expected band of amplicon ~ 260bp. Since there were many bands appeared in the gel, Dr. Zhang suggested to do PAGE size-selection to cut the 260bp band before doing PCR with the barcoded primers.
  • I repeated the amplification again in total volume 100ul by using the circularized DNA from 50, 52 and 54C annealing temperature and purified with Ampure beads (eluted volume 50ul). However, I used only the samples from 52 and 54C in PAGE size-selection and the next step.

File:ZhangLab 2 2011-10-11 00hr 03min-PAGE-SS-PGP1F.jpgFile:ZhangLab 2 2011-10-10 23hr 57min-PAGE-SS-PGP1iPS.jpg File:ZhangLab 2 2011-10-10 23hr 55min-PAGE-SS-H1.jpg

  • Resuspended each samplle with 12ul H2O.

Amplification with adapter primer

  • Reaction #1: CP-2-FA.IndSol (Indx =7, 45, and 76) & CP-2-RA.Sol
  • Reaction #2: Switch.CP-2-FA.Sol & Switch.CP-2-RA.IndSol (Indx = 7, 45, and 76)
  • I did the reaction #1 first to see if the amplification work.
Components 1 rxn 7.5 rxn mix
template 4.00 0.00
10uM CA-2-FA.Indx Sol 2.00 0.00
10uM CA-2-RA.Sol 2.00 15.00
2x KAPA MM 50.00 375.00
H2O 42.00 315.00
Total 100.00 750.00
  • Aliquot 94ul, add 4 ul template and 2ul Indx primer

Program
98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min

  • For both FA.IndSol or RA.IndSol, each sample used the same index number.

File:Reaction1.png

  • Reaction #2
Components 1 rxn 7.5 rxn mix
template 4.00 0.00
10uM Switch.CA-2-FA.Sol 2.00 15.00
10uM Switch.CA-2-RA.IndSol 2.00 0.00
2x KAPA MM 50.00 375.00
H2O 42.00 315.00
Total 100.00 750.00
  • Used the same PCR cycler as reaction #1

File:Reaction2.png

  • Purified PCR product with Ampure bead and eluted with 40ul EB buffer
  • Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel. I also loaded the size-selected DNA used as the template to see if I cut the right band.

File:ZhangLab 2 2011-10-11 05hr 37min-PQ-1.jpgFile:ZhangLab 2 2011-10-11 05hr 40min-PQ-2.jpg

  • 1 = PGP1 F 52C, 2 = PGP1F 54C, 3 = PGP1iPS 52C, 4 = PGP1iPS 54C, 5 = H1 52C, 6 = H1 54C