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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] == Plan for probe preparation for Matt's project == * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-2-27#Plan_for_probe_preparation_for_Matt.27s_project * 2013/03/06: received seed oligos from CustomArray * Seed oligo information ** 200 nt ** Total amount 4.95ug (61.93ng/ul in total volume 80ul in TE buffer) * Calculate MW of 200 nt probe = 200nt * 308.9D/bp + 79D = 61859 g/mol * 61.93ng/ul = 1.00114 ~ 1 uM or 1000 nM --> calculated from: 61.93(ng/ul)/61,859(g/mol) need to convert the unit to mol/L at the end * Aliquot 2ul of 61.93ng/ul oligos for PAGE verification & save original tube with Matt * Since the concentration is quit high, I will dilute to 100nM first (in 100ul) then dilute to 20uM (in 200ul) - To dilute from 1uM to 100nM volume 100ul: mix 10ul of 1uM oligos (61.93 ng/ul) with 90ul H2O - To dilute from 100nM to 20nM volume 200ul: mix 40ul of 100nM oligos with 160ul H2O === 3/07/2013 === ** After I diluted oligos to 100nM, I measured the oligo concentration by Nanodrop: 10.5 ng/ul (after dilute 10X, expected conc. ~ 6.1ng/ul) == Expansion PCR Test == * Test if the PCR works in small volume (50ul) by using LC Sciences oligoes as +ve control ** 1. NTC ** 2. +ve control 1nM LC Sciences oligos ** 3. 1nM 12k CustomArray oligos<br> {| {{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 3.5 rxn mix''' |- | 20nM seed oligoes||2.50||1nM||0.00 |- | eMIP_CA1_F (100uM)||0.20||400nM||0.70 |- | eMIP_CA1_R (100uM)||0.20||400nM||0.70 |- | 2x KAPA SYBG fast MM||25.00||1x||87.50 |- | H2O||22.10||||77.35 |- | Total||50.00||||175.00 |} * Aliquot 47.5ul, add 2.5ul of DNA template '''Program'''<br> 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold<br> == Result == * After I did 15 cycles of amplification, only reaction with LC sciences oligo works, but very small signal from 12k CustomArray oligos was observed (<500 fluorescent intensity, using Eppendorrf). This is possible that there are only 10-15% of the full-length in the oligo pools as Marcelo mentioned in email. [[File:2013_03_07_TestqPCR_12koligos_1nM.png| 600px]] == Repeat Expansion PCR Test == * I repeat amplification by increasing amount of oligos to 5X, 10X, and 20X, and used the 100nM oligos as the template. I am not sure if the high concentration of oligos containing the majority of truncated oligos will interfere the PCR that much or not * I also increase extension time to 45 sec even 30 sec is enough to synthesize 200nt DNA fragment, and increase the second step PCR to 15 cycles (total = 20 cycles). This is just to monitor the qPCR curve of each concentration. ** 1. NTC ** 2. +ve control 1nM LC Sciences oligos ** 3. 5nM 12k CustomArray oligos (5X, added 1.25ul of 100nM in 25ul reaction) ** 4. 10nM 12k CustomArray oligos (10X, added 2.5ul of 100nM in 25ul reaction) ** 5. 20nM 12k CustomArray oligos (20X, added 5ul of 100nM in 25ul reaction) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''5.5 rxn mix''' |- | 20nM seed oligoes||1.25||0.00 |- | eMIP_CA1_F (100uM)||0.10||0.55 |- | eMIP_CA1_R (100uM)||0.10||0.55 |- | 2x Kapa SYBG MM||12.50||68.75 |- | H2O||11.05||60.78 |- | Total||25.00||137.50 |} * Aliquot 23.75 '''Program'''<br> 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 45sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 45sec) x 15 -> 72C 2min -> 15C hold<br> == Results == [[File:2013_03_07_TestqPCR_12koligos_variedconc.png| 650px]] '''Ct values''' - +ve 1nM LC oligos = 14.26 - 5nM 12K oligos = 17.08 - 10nM 12K oligos = 16.36 - 20nM 12K oligos = 15.46 == PAGE verification == [[File:ZhangLab_2 2013-03-07 17hr 02min_TestExpansionPCR_variedconc.jpg| 300px]] O = 2ul of 12k oligos from original tubes * Note: I loaded 2.5ul of PCR products in TBU gel, so the size might be slightly different compared to in TBE gel. However, from the gel image, the size of amplicons is very close to 200bp fragment. I loaded PCR products in TBU gel becuase I didn't want to waste the TBU gel used for single strand oligos verification * From the image above, the amount of oligos in 2ul from original tubes was very high, but they all a smear. This is similar to what I observed when I ran the oligos from LC Sciences. * Since I used very high concentration of oligo templates, the smear DNA were observed. * For expansion PCR, I may not amplify as high as 20 cycles as in the test run. Need to decide the amount of oligos will be used for expansion PCR if I will amplify 15 cycles. <br> === 2013_03_08 === * Dr. Zhang suggested to do expansion PCR using 100nM oligos == Expansion PCR == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | 1 uM seed oligoes||20.00 |- | eMIP_CA1_F (100uM)||0.80 |- | eMIP_CA1_R (100uM)||0.80 |- | 2x KAPA SYBG fast MM||100.00 |- | H2O||78.40 |- | Total||200.00 |} * Spilt into 2 tubes '''Program'''<br> 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 9 -> 72C 2min -> 15C hold<br> [[File:2013_03_08_ExpansionPCR_12koligos_e.png| 550px]] * Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul * Measure DNA conc. with Nanodrop: 13.2 ng/ul or 103nM * M.W. of dsDNA = (# nucleotides x 607.4) + 157.9; 210bp * 607.4 Da/bp + 157.9 = 127711.9 g/mol ** 13.2ng/ul size 210bp = 103nM * Dilute 1st round amplicon to 10nM volume 400 ul (mix 38.83 ul of 103nM 1st round amplicons with 361.17 ul H2O): for using as the template for the future amplification * Perform production PCR == Production PCR == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''25x rxn mix''' |- | 1st round amplicon (10nM)||0.20||5.00 |- | eMIP_CA1_F (100uM)||0.40||10.00 |- | eMIP_CA1_R (100uM)||0.40||10.00 |- | 2x KAPA SYBG fast MM||50.00||1,250.00 |- | H2O||49.00||1,225.00 |- | Total||100.00||2,500.00 |} * Aliquot 100ul to 3 8-striptubes '''Program'''<br> 95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 14 -> 72C 2min -> 15C hold<br> [[File:2013_03_08_ProductionPCR_12koligos.png| 550px]] * Note: ** I increased extension time to 30sec) ** Number of cycle will be monitored * Perform EtOH precipitation of the 24 rxn PCR in 3x 15mL tube * 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH * Incubate at -80C for 30min --> centrifuge at 3,000 rpm for 30 min, wash with 75% EtOH * Resuspend with 100ul each tube * Re-purify with Qiaquick column (3 columns) and elute with 50ul EB buffer each --> total volume 150ul purified amplicons * Measure conc. with Nanodrop --> 89.6ng/ul, yield ~13.44ug. == Nicking endonuclease digestion == * Incubate ~3.2ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul (in NEBuffer2) at 37C for 1hr (4 x reactions) ** Each tube, 36ul 2nd round amplicons (89.6ng/ul), 20ul 10X NEBuffer2, 10ul Nt.AlwI, 134ul H2O * Heat inactivate enzyme at 80C for 20 min * Add 10ul Nb.BsrDI (10U/ul) at 65C for 1 hr * Before doing column purification, I ran the gel to check if DNA nicking work well by loading DNA before and after incubation with each nicking enzymes in TBU gel [[File:ZhangLab_2 2013-03-09 13hr 39min_Nicking.jpg| 150px]] I loaded 1ul of nicking enzyme reaction mix '''U'''= undigested '''Nt''' = Nt.AlwI '''Nt/Nb''' = Nt.AlwI & Nb.BsrDI * Note: >90% of the oligos were completely nicked by the two nicking enzymes. One thing I am concerned is the size of probes is quite long, so the 210, 190 and 170nt stay very close in 6% TBU gel. I may need to run a little longer for 5 or 10 minute to get a better resolution. * 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 --> 37.8 ng/ul total volume 6*50 = 300ul, yields = 11.34 ug (recovery rate ~ 84.4%) * I did PAGE size-selection by loading ~1.8ug (to purify in 6x 6% TBU gels and prevent overloaded because the bands are very close). Since the 10bp ladder resolution around 150-higher is not very good, I loaded nicked DNA as marker in small well since I did PAGE verification before as shown in the image above. [[File:ZhangLab_2 2013-03-09 19hr 22min_PAGE_SS-1e.png| 400px]] [[File:ZhangLab_2 2013-03-09 21hr 01min_PAGE_SS-2.jpg| 410px]] [[File:ZhangLab_2 2013-03-09 21hr 01min_PAGE_SS-3.jpg| 400px]] * EtOH precipitate and resuspend functional probes in total volume 260ul. * Perform PAGE quantification in 6% TBU gel (mix 2ul of probes in total volume 100ul and load different volume -> 2, 4, 6, 8, and 10ul) * Actual loading volume is 0.04, 0.08, 0.12, 0.16 and 0.2 ul [[File:2013_03_10_PQ_12K_InsituProbes.png| 300px]] * From the gel image, it seemed like I loaded too small volume and the intensities of ladder were not very reliable. The reliable volume is ~ 0.16 and 0.20 ul of actual volume of the probes ~3.7ng/ul. So the total yields ~ 3.7ng/ul * 260ul ~ 962ng. * However, I might re-quantify or do Qubit ssDNA assay to confirm concentration before start capturing * Note: Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul (by column purification I lose ~10-15% probes) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-12
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