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= RRBS library preparation of colon tumor tissue samples from Biochain = * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * References ** scRRBS: [http://genome.cshlp.org/content/23/12/2126.full.pdf] ** Standard RRBS (Nature Protocols): [http://www.nature.com/nprot/journal/v6/n4/pdf/nprot.2010.190.pdf] ** Gel-free RRBS (multiplexed): [http://genomebiology.com/content/pdf/gb-2012-13-10-r92.pdf] ==== Sample list and details ==== * Sample type: genomic DNA from primary tumor tissue ** Colon tumor tissue, frozen (CTT-frozen): re-quantified concentration with Qubit dsDNA HS assay ** Colon tumor tissue, FFPE (CTT-FFPE) * 2014-06-01, I added TE buffer to make conc. to 50ng/ul (total volume 40ul) based on original conc. and re-measured conc. with Qubit dsDNA HS assay again (1ul for the assay). * Dr. Zhang suggested to do two replicates for each level * I will spike in 0.5% of unmethylated lambda DNA to check bisulfite conversion rate. :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Original conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Estimate volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Dilute to 50ng/ul (total volume)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Add TE buffer (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Qubit conc. (ng/ul)''' |- | CTT-Frozen||580.00||3.45||40.00||36.55||52.3 |- | CTT-FFPE||76.00||26.32||40.00||13.68||64.8 |} ==== Library preparation ==== * Low-input RRBS (5ng) * Standard RRBS (100ng) == Experimental procedures == * I firstly dilute CTT gDNA to 2ng/ul volume 50ul :{| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Stock volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''TE (ul)''' |- | CTT-frozen||52.30||1.91||48.09 |- | CTT-FFPE||64.80||1.54||48.46 |} * For 5ng input, I will add 25pg of unmethylated lambda DNA (add 2.08ul of stock 12pg/ul) * For 100ng input, I will add 0.5ng of unmethylated lambda DNA (add 0.5ul of stock 1ng/ul) === 1) DNA fragmentation with ''Msp''I === ====1.1) Low-input RRBS ''Msp''I digestion set up ==== :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-5ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-5ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''5ng NTC''' |- |} :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 5ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Buffer''' | align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''12pg/ul unmeth-lambda DNA''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | 1.1 CTT-frozen||2.00||2.5||2.00||1.00||2.08||10.42||18.00 |- | 1.2 CTT-FFPE||2.00||2.5||2.00||1.00||2.08||10.42||18.00 |- | 1.3 NTC||0.00||2.5||2.00||1.00||2.08||10.42||18.00 |} :<u>'''''Msp''I reaction mix I'''</u> :{| {{table}} border = 1 | align="center" style="width:150px;background:#f0f0f0;"|'''Components''' | align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix''' |- | MspI (10U/ul)||1.00||5.50 |- | 10X Tango Buffer||2.00||11.00 |- | 12pg/ul unmeth-lambda DNA||2.08||11.44 |- | H2O||10.42||57.31 |- | Total||15.50|| |} :- Aliquot 15.50ul to each tube :- Add 2.5ul of diluted CTT DNA to each tube :- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down ====1.2) STD RRBS ''Msp''I digestion set up ==== :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC''' |- |} :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10X Tango Fuffer''' | align="center" style="width:80px;background:#f0f0f0;"|'''MspI (10U/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to adjust volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''1ng/ul unmeth-lambda DNA''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O in MspI mix(ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | 1.1 CTT-frozen||52.30||1.91||2.00||1.00||5.09||0.50||7.50||18.00 |- | 1.2 CTT-FFPE||64.80||1.54||2.00||1.00||5.46||0.50||7.50||18.00 |- | 1.3 NTC||0.00||0.00||2.00||1.00||7.00||0.50||7.50||18.00 |} :<u>'''''Msp''I reaction mix II'''</u> :{| {{table}} border = 1 | align="center" style="width:150px;background:#f0f0f0;"|'''Components''' | align="center" style="width:90px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:90px;background:#f0f0f0;"|'''5.5 rxn mix''' |- | MspI (10U/ul)||1.00||5.50 |- | 10X Tango Buffer||2.00||11.00 |- | 1ng/ul unmeth-lambda DNA||0.5||2.75 |- | H2O||7.50||41.25 |- | Total||11.00|| |} :- Add H2O to adjust volume as table above :- Aliquot 11ul to each tube :- Add CTT DNA to each tube following the number in table above :- Mix by gentle pulse-vortexting for 10x (put reaction tube on PCR rack) and spin down --------------------------------------------------------------------------- :- Incubate at 37C for 3hr :- Heat inactivate at 65C for 20min === 3) End-repair/dA-tailing === '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - To prevent contamination to enzyme tube by multiple time pipetting, I aliquot 11ul each of <u>Klenow fragment, exo-</u> and <u>dA:dC:dG mix</u> into PCR tube.<br> - dA:dC:dG solution was prepared in the concentration (20mM:2mM:2mM). Final concentration in 20ul reaction is 1mM:0.1mM:0.1mM. :- Add 2ul of dA:dC:dG/Klenow fragment exo- to each tube :- Spin down the tube :- Mix by gentle pulse-vortexting for 10x :- Spin down the tube :- Incubate at 30C for 20min (for gap-filling) --> 37C for 20min (for extra dA-tailing) <u>no heat lid will help to protect Klenow fragment, exo</u> :- Heat inactivate enzyme at 75C for 10min :- Set program to hold at 4C :- Spin down the tube before continuing to next step === Methylated adaptor ligation === - To avoid the overlapping of indexes to some scRRBS libraries that may need to sequence in the same run (HiSeq Rapid run has only 2 lanes), I will not use index 2, 4, 5, and 12<br> - I will use 1ul of 1:10 diluted TruSeq adaptors for both low-input RRBS and STD RRBS.<br> '''Index list'''<br> {| {{table}}class = wikitable | align="center" style="width:80px;background:#f0f0f0;"|'''CCT-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''5ng NTC''' |- | Ind_9||Ind_10||Ind_11||Ind_13||Ind_14 |} {| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC''' |- | Ind_15||Ind_16||Ind_18||Ind_19||Ind_20 |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix - Prepare ligation reaction mix :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''11x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||5.50 |- | HC T4 DNA ligase (30units/ul)||1.00||11.00 |- | 10mM ATP ||1.25||13.75 |- | H2O||1.25||13.75 |- | Total||24.00||44.00 |} :- Add 1ul of diluted methylated adapter :- Add 4ul of ligation reaction mix :- Spin down the tube :- Mix by gentle pulse-vortexting for 10x :- Spin down the tube :- ''Incubate at 16C for 18h (no heat lid)'' :- ''Heat inactivate at 65C for 20min'' '''2014-06-03, continued'''<br> ==== Low-input RRBS ==== :- Heat inactivate at 65C, 20min :- Wait for STD RRBS sample for AMPure bead purification ==== STD RRBS ==== - AMPure bead purification with 2X volume of the bead :- Mix 50ul of AMPure beads with 25ul ligated DNA. Mix by pipetting 10x :- Sit for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 160ul freshly prepared 75% EtOH :- Dry the bead for 3-5min :- Resuspend with 27 H2O :- Transfer to new tube for bisulfite conversion. 25ul will be used for bisulfite conversion. === 5) Bisulfite conversion === * I performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 31ul elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare 1 tubes of CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850) :- Add125ul of complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube) :- Mix by pipetting 10X with multi-channel pipette :- Spin down the plate at 2,000rpm for 1min :- Incubate following below program ::- 98°C for 10 minutes (DNA denaturation) ::- 64°C for 2.5 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA<br> - For 10.2 rxn, I mixed 6.12mL of Binding Buffer with 10.2ul of 10ng/ul tRNA (this actually for low-input, but I think it should be fine) : - Add 601ul of Binding Buffer/tRNA mix to the column :- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. I rinse the well with small amount of Binding Buffer to transfer DNA to the column as much as possible :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash with 100ul Wash buffer :- Spin down 14,000rpm for 30sec :- ''Incubate with 200ul of Desulphonation Buffer for 18min'' :- Spin down 14,000rpm for 30sec :- Wash column with 200ul Wash Buffer. :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash the column with 200ul Wash Buffer. :- Spin down 14,000rpm for 2min :- Elute converted DNA with warm (~60C) 31ul Elution Buffer. Incubate column with Elution Buffer at room temp for 2min. :- Spin down at 14,000rmp for 1min. This should have ~30ul DNA left for PCR ---- ---- = Sensitivity test of MONOD V1N3 BSPP capture with pre-amplification = * There are many things need to concern about BSPP capture of serum/plasma samples for this project. ** DNA amount from plasma is very low in the range of 5-10ng from 1mL plasma. ** Serum samples showed higher amount than plasma, but Dr. Zhang suggest that the high amount is from the lysis of white blood cell. ** If we use serum samples, we need to increase the sensitivity of cancer DNA detection. ** If we use plasma sample, we need to do some pre-amplification to amplify <10ng of cell-free DNA by ~10-fold for padlock capture. * The amount that Dr. Zhang meant is amount of extracted DNA not after bisulfite conversion. I have done some experiment to test the recovery rate of bisulfite conversion at different input amounts. ==== Bisulfite conversion recovery rate #1 ==== * Note: The resulting BIS-DNAs were used for sensitivity test experiment {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:100px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield in 15ul (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Input DNA (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|''% Recovery'' |- | JK-1||15.2||ng/mL||1||200||3.03||45.45||bgcolor="Aqua"|100||bgcolor="Aqua"|45.45 |- | JK-2||19.1||ng/mL||1||200||3.82||57.30||bgcolor="Aqua"|100||bgcolor="Aqua"|57.30 |- | JK-3||13.3||ng/mL||1||200||2.66||39.90||bgcolor="Aqua"|100||bgcolor="Aqua"|39.90 |- | JK-4||41||ng/mL||1||200||8.21||123.15||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|61.58 |- | JK-5||36.6||ng/mL||1||200||7.31||109.65||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|54.83 |- | JK-6||39.4||ng/mL||1||200||7.88||118.20||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|59.10 |- | JK-7||133||ng/mL||1||200||26.6||399.00||bgcolor="royalBlue"|600||bgcolor="royalBlue"|66.50 |- | JK-8||131||ng/mL||1||200||26.2||393.00||bgcolor="royalBlue"|600||bgcolor="royalBlue"|65.50 |} ==== Bisulfite conversion recovery rate #2 ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:100px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield in 9ul (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Input DNA (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''% Recovery''' |- | JK-1||3.8||ng/mL||1||200||0.760||6.84||bgcolor="lightcyan"|25||bgcolor="lightcyan"|27.36 |- | JK-2||4.9||ng/mL||1||200||0.980||8.82||bgcolor="lightcyan"|25||bgcolor="lightcyan"|35.28 |- | JK-3||2.5||ng/mL||1||200||0.500||4.50||bgcolor="lightcyan"|25||bgcolor="lightcyan"|18.00 |- | JK-4||5.11||ng/mL||1||200||1.022||9.20||bgcolor="lightcyan"|25||bgcolor="lightcyan"|36.79 |- | JK-5||10.8||ng/mL||1||200||2.160||19.44||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|38.88 |- | JK-6||9.06||ng/mL||1||200||1.812||16.31||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|32.62 |- | JK-7||12.3||ng/mL||1||200||2.460||22.14||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|44.28 |- | JK-8||11.2||ng/mL||1||200||2.240||20.16||bgcolor="PaleTurquoise"|50||bgcolor="PaleTurquoise"|40.32 |- | JK-9||25||ng/mL||1||200||5.000||45.00||bgcolor="aqua"|100||bgcolor="aqua"|45.00 |- | JK-10||26.7||ng/mL||1||200||5.340||48.06||bgcolor="aqua"|100||bgcolor="aqua"|48.06 |- | JK-11||28.4||ng/mL||1||200||5.680||51.12||bgcolor="aqua"|100||bgcolor="aqua"|51.12 |- | JK-12||24.3||ng/mL||1||200||4.860||43.74||bgcolor="aqua"|100||bgcolor="aqua"|43.74 |- | JK-13||90.1||ng/mL||1||200||18.020||162.18||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|81.09 |- | JK-14||89||ng/mL||1||200||17.800||160.20||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|80.10 |- | JK-15||79.7||ng/mL||1||200||15.940||143.46||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|71.73 |- | JK-16||85.4||ng/mL||1||200||17.080||153.72||bgcolor="DeepSkyBlue"|200||bgcolor="DeepSkyBlue"|76.86 |} * Input 200ng had too high recovery rate compare to previous experiment. * As Dr. Zhang mentioned about pre-amplification before BSPP capture, I wanted to do experiment to test if I can get the capture work on pre-amplified DNa again. * I used BIS-DNA of cancer sample #2 (CANC-2) because I had no time to prepare more BIS-DNA. * I set up experiment by including 5ng, 10ng, 25ng and 50ng of BIS-DNA in amplification in total 10ul reaction with the following condition (will be added in very details) ** Mix BIS-DNA with 2uM N9 random primers and 1X Reaction buffer for Klenow, exo- ** Heat at 94C, 3min --> 30C (ramp 0.2C/sec) 3min --> add dNTP & Klenow, exo- mix (400uM dNTP, 2.5U of Klenow,exo---> 30C, 1h --> heat inactivate at 75C, 20min ---- == Pre-amplification set up == {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''bis-CANC_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount required (ng)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume for XX ng (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:70px;background:paleturquoise;"|'''50uM N9''' | align="center" style="width:70px;background:paleturquoise;"|'''10X reaction buffer''' | align="center" style="width:70px;background:#f0f0f0;"|'''Total''' |- | 5a||5.00||5.00||1.00||4.80||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 5a||5.00||5.00||1.00||4.80||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 10a||5.00||10.00||2.00||3.80||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 10a||5.00||10.00||2.00||3.80||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 25a||21.70||25.00||1.15||4.65||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 25a||21.70||25.00||1.15||4.65||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 50a||21.70||50.00||2.30||3.50||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | 50a||21.70||50.00||2.30||3.50||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |- | NTC-a||0.00||0.00||0.00||5.80||bgcolor="PaleTurquoise"|0.40||bgcolor="PaleTurquoise"|1.00||7.20 |} <u>'''N9 and reaction buffer for Klenow, exo- mix'''</u> {| {{table}} | align="center" style="width:120px;background:#f0f0f0;"|'''N9 & Buffer Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''15X rxn''' |- | 10X Reaction Buffer||1.00||15.00 |- | 50uM N9||0.40||6.00 |- | Total||1.40|| |} <u>'''Klenow, exo- & dNTP mix'''</u> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Klenow, exo- & dNTP mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''12x rxn''' |- | 10mM dNTP Mix||0.40||4.80 |- | Klenow Fragment, exo - (5U/ul)||0.50||6.00 |- | H2O||1.90||22.80 |- | Total||2.80|| |} * Final conc. of dNTP in the reaction = 0.4mM or 400uM * Final conc. of N9 primers in the reaction = 2uM * Total amount of Klenow, exo- in 10ul = 2.5 units :- Mix DNA with H2O and 1.4ul of N9/reaction buffer mix :- Heat at 94C, 3min --> 30C (ramp 0.2C/sec) 3min --> add 2.8ul of dNTP & Klenow, exo- mix (400uM dNTP, 2.5U of Klenow,exo- in the hood)---> 30C, 1h --> heat inactivate at 75C, 20min :- Purified with Zymo DNA Clean & Concentrator-5 column (I added 60ul of binding buffer, 6x volume) --> elute 8ul <br> :- I used 5ul for MONOD V1N3 capture. The left over DNA in the tube ~1-1.5ul were loaded in TBE gel to verify if the amplification work well. :- ''I loaded 50ng of BIS-DNA before amplification in one lane for comparison'' :- ''I also load 1ul of Illumina amplified DNA as control'' <br> :[[File:ZhangLab_2 2014-06-02 23hr 09min_low-amp_TBE-verify.jpg| 500px]] :- <u>The numbers showed input BIS-DNA for amplification.</u> * From the gel image, it showed that amplification in all input DNA worked well as no amplification in NTC and un-amplified BIS-DNA. I was wondering where is the top band of BIS-DNA. I am concern if it could be purified with Zymo column (DNA Clean & Concentrator™-5 - Capped Columns). * The pattern of amplified DNA looked similar to Illumina's amplified DNA. == BSPP capture set up == * Since I expected that the amount of template would increase after amplification, I roughly estimate it to be ~10X--> after column purification, I assume 5X left over. * I therefor increase the amount of BSPP to 5X based on 1000:1 probe to target ratio. * Perform BSPP capture the same volume and incubation time as experiment on 2014-06-01: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-6-1]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Input BIS-DNA (ng) in pre-amp''' | align="center" style="width:120px;background:#f0f0f0;"|'''Assume 5X amount after pre-amp (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Probe required (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''V1N3 conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''V1N3 volume (ul)''' |- | 5||25||1.10||0.73||1.51 |- | 10||50||2.20||0.73||3.01 |- | 25||125||5.50||6.75||0.81 |- | 50||250||11.00||6.75||1.63 |} {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''strip 1''' | align="center" style="width:60px;background:#f0f0f0;"|'''5a''' | align="center" style="width:60px;background:#f0f0f0;"|'''5a''' | align="center" style="width:60px;background:#f0f0f0;"|'''10a''' | align="center" style="width:60px;background:#f0f0f0;"|'''10a''' | align="center" style="width:60px;background:#f0f0f0;"|'''25a''' | align="center" style="width:60px;background:#f0f0f0;"|'''25a''' | align="center" style="width:60px;background:#f0f0f0;"|'''50a''' | align="center" style="width:60px;background:#f0f0f0;"|'''50a''' |- | Volume (ul)||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00 |- | H2O (ul)||2.49||2.49||0.99||0.99||3.19||3.19||2.37||2.37 |- | V1N3 amount (ng)||1.10||1.10||2.20||2.20||5.50||5.50||11.00||11.00 |- | V1N3 conc. (ng/ul)||0.73||0.73||0.73||0.73||6.75||6.75||6.75||6.75 |- | V1N3 volume (ul)||1.51||1.51||3.01||3.01||0.81||0.81||1.63||1.63 |- | 10X Ampligase Buffer||1.00||1.00||1.00||1.00||1.00||1.00||1.00||1.00 |- | Total||10.00||10.00||10.00||10.00||10.00||10.00||10.00||10.00 |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 5h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold. == Amplification == * I used 10ul of captured DNA in total 75ul PCR reaction. :{| {{table}} | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:60px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:60px;background:#f0f0f0;"|'''13.5 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.50||20.25 |- | 10uM AmpR6.3.IndX||1.50||0.00 |- | 2X KAPA SYBR MM||37.50||506.25 |- | H2O||24.50||330.75 |- | Total||75.00|| |} :- Aliquot 63.5ul + 1.5ul AmpR6.3 IndX + 10ul template :- Set cycle number maximum at 27X '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min {| {{table}} border = 1 | align="center" style="width:150px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | 1000:1_5ng-preamp_1||Ind_1 |- | 1000:1_5ng-preamp_2||Ind_2 |- | 1000:1_10ng-preamp_1||Ind_3 |- | 1000:1_10ng-preamp_2||Ind_4 |- | 1000:1_25ng-preamp_1||Ind_5 |- | 1000:1_25ng-preamp_2||Ind_6 |- | 1000:1_50ng-preamp_1||Ind_7 |- | 1000:1_50ng-preamp_2||Ind_8 |} * Loaded 4ul of PCR products in 6% TBE gel (the same volume as 2014-06-01) [[File:ZhangLab_2 2014-06-06 14hr 13min_V1N3_CANC2-5-50ng-pream.jpg| 400px]] ===== Compare the qPCR results with experiment on 2014-06-01 with no pre-amplification before BSPP capture ===== * Note that, I only focus on 5 and 10ng input '''no pre-amplification''' [[File:2014-06-03_5ng-noamp.png| 400px]] [[File:2014-06-03_10ng-noamp.png| 400px]] '''Pre-amplification''' [[File:2014-06-06_5ng-preamp.png| 400px]] [[File:2014-06-06_10ng-preamp.png| 400px]] * The qPCR curves between no-amp and pre-amp may not be able to use for comparison as there is more background in pre-amp capture. ===== Compare the gel image ===== '''no pre-amplification''' [[File:ZhangLab_2 2014-06-03 16hr 20min_V1N3_1000to1.jpg| 420px]] '''Pre-amplification''' [[File:ZhangLab_2 2014-06-06 14hr 13min_V1N3_CANC2-5-50ng-pream.jpg| 420px]] * From the gel images, it looked like the intensity of BSPP capture with pre-amplification is higher than no pre-amplification, especially 10ng input. For 5ng, the reactions with pre-amp seemed to be a little brighter than no-amp. * However, there is more background in pre-amp capture. I guess this might be decrease with 1-2 less cycle numbers. * I should have done these experiments side by side to confirm that a smear band did not come from the original BIS-DNA template as without amplification, the capture worked. '''Gel image of BSPP capture of pre-amplified DNA without column purification before ecapture'''<br> '''experiment on 2014-04-10''' [[File:ZhangLab_2 2014-04-10 18hr 50min_GP1-V4-gel2_ee.png| 450px]]<br>
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