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== 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.
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