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= MONOD_V3_12k probe preparation = [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br> '''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 == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''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 == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''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 ==== : - 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 ==== * I reduce digestion volume to 120ul as I reduce DNA amount to ~10ug <u>'''1x rxn'''</u> <u>'''4x rxn'''</u> 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 ==== * I reduce digestion volume to 120ul as I reduce DNA amount to ~10ug <u>'''1x rxn'''</u> <u>'''2.5x rxn'''</u> 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 <u>'''1x rxn'''</u> <u>'''2.6x rxn'''</u> 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 ==== : - 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 ====
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