Thursday 04/11/13

Posted on April 11, 2013 by admin

Oligo Paint Probe synthesis

  • Test PCRs at 1 mL scale:
  1. Assymetric y-PCR, Y-adapter, no Y-L primer + 1uL of EtOH extracted Y PCR product at 85 ng/uL
  2. Assymetric b-PCR, B-adapter + 1 uL of EtOH extracted B PCR product at 63 ng/uL
  3. PPT-protected primer, y-PCR + 1uL of EtOH extracted Y PCR product at 85 ng/uL
  4. PPT-control primer, y-PCR + 1uL of EtOH extracted Y PCR product at 85 ng/uL
  5. PPT-protected primer, b-PCR + 1 uL of EtOH extracted B PCR product at 63 ng/uL
  6. PPT-control primer, b-PCR + 1 uL of EtOH extracted B PCR product at 63 ng/uL
  7. y-PCR from orig. library, old + 10uL 10mM new nucleotide + 1uL of 15 ng/uL emPCR library
  8. y-PCR from orig. library, lower temp cycles + 1uL of 15 ng/uL emPCR library
  9. y-PCR from orig library, 4x primer + 1uL of 15 ng/uL emPCR library

Setup

  • 10 mL master mix of enzyme, buffer and nucleotides
  • 120 uL per PCR tube
  • running all but #8 together on original PCR program.
  • 1 hr 10 min to set up as individual PCR reactions.

Results Qubit, [Nanodrop]

  1. y asym = 57.1 ug/mL in ~ 1mL. (N ug)/(116660)10^3 = N nmol dsDNA 116 bp probe. = 0.75 nmol (added 1 nmol primer) [510 ng/uL]
  2. b asym = 37 ug/mL –> 0.48 nmol probe [506 ng/uL]
  3. y ppt prot = 56.8 ug/mL –> 0.74 nmol [488 ng/uL]
  4. y ppt cntrl = 54.4 ug/mL –> 0.71 nmol [452 ng/uL]
  5. B ppt prot = 58.8 ug/mL –> 0.77 nmol [440 ng/uL]
  6. B ppt cntrl = 60.5 ug/mL –> 0.79 nmol [436 ng/uL]
  7. Y NTP 351 ng/mL => 23.4 ug/mL –> 0.31 nmol [846 ng/uL]
  8. Y temp 318 ng/mL => 21.2 ug/mL –> 0.28 nmol
  9. Y 2xPrimer = 38 ug/mL –> 0.50 nmol [522 ng/uL]
  10. B from 10 mL 63 ug/mL [3446 ng/uL]
  11. Y from 10 mL 85 ug/mL [1698 ng/uL]

Conclusions

  • Primer is limiting in initial, inefficient reaction from emPCR library
  • y and b asym PCR have similar dsDNA yields to the other reactions, probably excess R primer available?

PCR cleanup

T7 Exonuclease digestion

  • samples
  1. B ppt prot (1/2), no PCR cleanup
  2. B ppt prot (1/2) with PCR cleanup
  3. Y emPCR Temp, (1/2), no PCR cleanup
  4. B no ppt control, with PCR cleanup
  5. Y ppt prot, with PCR cleanup
  6. Y no ppt prot, with PCR cleanup
  • procedure
  1. 0.15 mM DNA digested in 30 min by 1 unit (NEB T7 page)
  2. I have ~.75 nmol in 150 uL (hopefully). = 5,000 nmols/L = 5 mM. 10 units in 50 uL (max amount of glyercol for restriction enzymes) should digest at most 1.5 mM. Lets do 5 50 uL reactions. And there seems to be no reason I can find to keep the total reaction volume small (only warnings are against too small, not too large), so let’s make it 1 250 uL reaction. With 50 units of T7. (10 units per uL).
  3. 5 uL of T7-Exonuclease + 25 uL NEB buffer 4 + 150 uL eluted DNA + 70 uL ddH2O
  4. 5 uL of T7-Exonuclease + 50 uL NEB buffer 4 + 400 uL eluted DNA
  5. Incubated 1 hr
  6. Run large scale Clean-up / Concentration.

Test gel

  • Order:
  • 10 uL Yem,
  • 10 uL Y 10mL EtOH extracted
  • 10 uL B 10mL EtOH extracted
  • 5 uL Assym Yellow PCR
  • 5 uL Assym B PCR
  • ULR diluted 1:20
  • Running on 5% gel. 100V 3min start, 200V 30 min run.

Nb.BsrDI Digestion

  • Y DNA from 10 mL PCR round 1
  • B DNA from 10 mL PCR round 1

Check samples

  • Can’t get into Bauer Core to use Qubit. Check tomorrow.
  • Many extra bands on gel :(. Concentration is too high. Gel needs to be run longer (running now).

Review writing

  • working on discussion section

Other

  • track ring – delivered
  • track passport – to be delivered April 12th
  • Meeting with Jorge
  • Cells for Joe. — Cells plated in regular petri dish, not cell culture dishes. Not adherent! Replating in cell culture dishes.
  • Banff Invites. Sent out next 7 invites, updated invitee list.
  • Sent list of confirmed participants to Banff
  • Request from researcher at Biophysics Unit of Bizkaia for DaoSTORM support. Directed to Hazen.
  • 3D DaoSTORM license includes permission to use/modify/distribute/sublicense, as long as original license is included in all copies of the software.

Review

  • working on final discussion.
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