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