Noi/NOTES/2014-3-26

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MONOD_V3_12k probe preparation[edit]

[Link to calendar]
2013-03-26

- Received oligos from CustomArray
Oligo information
- Design: BSPP_Design-5-17-2013 --> I would name BSPP_Blueprint for future experiment NEED TO FIXED!!!
- Length 146 mer (MW = 146 * 330Da/bp = 48,180 Da or g/mole
- Cocn. 30.35ng/ul = 629 nM (30.35ng/ul/ 48,180 g/mole)
- Volume 80ul in TE buffer (The actual volume is smaller than 80ul

Expansion PCR[edit]

Components Volume (ul) Final conc. Volume for 1.5 rxn mix
Seed oligo (629nM) 15.90 100nM 23.85
AP1V61U (100uM) 0.40 400nM 0.60
AP2V6 (100uM) 0.40 400nM 0.60
2x KAPA SYBG fast MM 50.00 1x 75.00
H2O 33.30 49.95
Total 100.00 150.00
  • Aliquot 75ul to 2 of WW tubes

Program

95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold

* I stopped reaction at 13 cycles
- Purified 1st round amplicon in 2 QIAquick column and elute with EB buffer 50ul each (Final volume left ~95ul)
- N.D. = 7.35ng/ul
MW of dsDNA 146mer = 146*660 = 96360g/mole
7.35ng/ul = 14.7ng/ul/96360g/mole/10^6 = 152.55nM
- Dilute 1st round amplicon to 20nM by mixing 90ul of 14.7ng/ul 1st round amplicons with 596.48ul TE buffer
  • I 20nM 1st round amplicons as template instead of 10nM since oligo pools from CustomArray has very high background even after expansion PCR. I use the same volume for probe production.

Production PCR[edit]

Components 1 rxn 50x rxn mix
1st round amplicon (20nM) 0.20 10.00
AP1V61U (100uM) 0.40 20.00
AP2V6 (100uM) 0.40 20.00
2x KAPA SYBG fast MM 50.00 2500.00
H2O 49.00 2450.00
Total 100.00 5000.00
  • Aliquot 100ul to each well in half of 96-well plate
Program

95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold
* I stopped reaction at 14 cycles

EtOH precipitation[edit]

- Do EtOH precipitation of 48 rxn PCR in 6x 15mL tube
- 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH
- Incubate at -80C O/N
- Centrifuge at 3,000 rpm for 30min. Discard spnt.
- Wash DNA pellet by adding 800ul of ice cold 80% EtOH, transfer DNA pellet to 1.5mL Lobind tube, rinse 15mL tube with 200ul EtOH and transfer to 1.5mL tube.
- Discard spnt.
- Resuspend each tube with 100ul H2O, purified with 6X QIAGEN column, eluted 62ul each tube. Total volume left 360ul.
- N.D. dsDNA = 103.9ng/ul
- Yield = 360ul * 103.9ng/ul = 37.4ug.

Lambda Exonuclease digestion[edit]

  • I reduce digestion volume to 120ul as I reduce DNA amount to ~10ug
                                 1x rxn          4x rxn
dsDNA (103.9ng/ul)                90ul           360ul     (DNA amount /tube = 9.35ug)
10X Lamda Exo Buffer              12ul            48ul
Lambda exonuclease                 9ul            36ul
H2O                                9ul            36ul
---------------------------------------------------------
Total                            120ul           ALiquot 120ulx4 tubes
- Incubate at 37C for 1.5h
- Purify ssamplicon with 4x ssDNA/RNA clean & concentrator column, ZymoResearch
- Follow protocol provided with the kit (for volume 120ul)
- Elute each column with 40ul H2O. Total volume left = 150ul)
- N.D. ssDNA = 85.3ng/ul
- Yields = 150ul * 85.3ng/ul = 12.8ug

Removal of amplification adaptors[edit]

  • I reduce digestion volume to 120ul as I reduce DNA amount to ~10ug
                                 1x rxn          2.5x rxn
ssDNA (85.3ng/ul)                 60ul           150ul     (DNA amount /tube = 5.11ug)
USER                               5ul          12.5ul
H2O                               15ul          37.5ul
--------------------------------------------------------
Total                             80ul          Aliquot 80ulx2 and 40ulx1
- Incubate at 37C for 2h
                                 1x rxn          2.6x rxn
10x DpnII buffer                  10ul            26ul
100uM RE-DpnII-V6 guide oligos     5ul            13ul
--------------------------------------------------------
Total                             15ul
- Add 15ul and 7.5ul of 10X DpnII buffer and RE-DpnII-V6 guide oligos to 80ul and 40ul reaction tubes, respectively.
- Mix well and incubate at 94C for 3min -> 37 C 3min
- Add 5ul or 2.5ul of DpnII to 80ul and 40ul reaction tubes, respectively.
- Mix by pipetting 10 times on thermocycler
- Incubate at 37C for 3h. Heat inactivate at 75C for 20min
- Purify USER/DpnII digested ssamplicon with 3x ssDNA/RNA clean & concentrator column, ZymoResearch
- Follow protocol provided with the kit (for volume 120ul)
- Elute each column with 62ul H2O. Total volume left = 180ul)
- N.D. ssDNA = 44.5ng/ul
- Yields = 180ul * 44.5ng/ul = 8ug

PAGE size-selection[edit]

- Perform PAGE size-selection in 3X 2D-well TBU gel, by loading 60ul of 44.5ng/ul ssDNA, ~2.67ug DNA/gel
- Purified in 9X 1.5 mL lo-bind tube and resuspend each tube with 11ul H2O (total volume ~99ul--> left over ~95ul after quantification)
- Quantify working probe concentration by Qubit ssDNA assay (use 1ul in 200ul reaction, 200x dilution)
- Qubit ssDNA conc. 12.2 ng/ul --> yield = 95ul * 12.2ng/ul = 1,159ng

PAGE verification in 6% TBU gel[edit]