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

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* Note: The digestion was not very complete since I still see the partial digested band of ~ 100 bp. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high. However in this experiment I have no time to optimize and I think that the expected band (~70nt) is a lot brighter than the incomplete digested band, so I decided to go PAGE size-selection to purify the 70nt band in 6% tBU gel.  
* Note: The digestion was not very complete since I still see the partial digested band of ~ 100 bp. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high. However in this experiment I have no time to optimize and I think that the expected band (~70nt) is a lot brighter than the incomplete digested band, so I decided to go 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.
* 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.jpg| 300px]][[File:ZhangLab_2 2011-10-08 00hr 15min_PAGE-SS-2.jpg| 300px]]<br>
[[File:ZhangLab_2 2011-10-08 00hr 11min_PAGE-SS-1.jpg| 300px]][[File:ZhangLab_2 2011-10-08 00hr 15min_PAGE-SS-2.jpg| 320px]]<br>
* I used the old TBU gel prepared 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.
* I used the old TBU gel prepared 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.

Revision as of 16:31, 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 probes 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
  • 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 4 15mL tube
    • amplified amplicon 800ul+80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH
    • incubate at -80C for 30min --> centrifuge at 3000 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
  • 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 ~ 100 bp. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high. However in this experiment I have no time to optimize and I think that the expected band (~70nt) is a lot brighter than the incomplete digested band, so I decided to go 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 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.