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AlanFung:LabNotes/CTCF/2011-8-19
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==Preparation for Sanger Sequencing== ===Objective=== *Clones grew well after overnight incubation, prepare clones for sanger sequencing. (a bit overgrown and most of the clones are blue in color, we can either plate a smaller volume of tranformation mixture or dilute 10 fold with soc before plating) ==Protocol== ===PCR amplification and preparation for sanger sequencing=== *Add 12.5ul of 100ul of F and R primer and add 475ul sterile h2o to make 2.5uM primer mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | DNA||Pick one colony per well||Do not add to MM |- | H2O||25||205 |- | M13-21 F+R Primer (2.5uM each)||8||65.6 |- | 2X Taq MM||15||123 |- | Total||48||393.6 |} *Add 48ul MM to each colony 94C 10 min to break up the cells 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever ===PAGE=== [[File:ZhangLab_2 2011-08-19 16hr 58min.jpg|500px]] ==Results== *For some reason, no amplicons were formed, I suspect the ligation failed ==Troubleshoot== *Either the polymerase or the pcr condition is not optimised, I will test both parameters with the control reactions to test the activity of the polymerase ==Producing the Control PCR Product== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per Reaction''' |- | Control DNA template (100ng)||1 |- | M13 F+R (2.5uM each)||8 |- | Sterile water||16 |- | TagMM||25 |- | Total Volume||50 |} 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever ===Page Gel Analysis=== [[File:ZhangLab_2 2011-08-23 18hr 26min.jpg|500px]] ==Results== *No amplicons seen on page gel, I guess the polymerase has gone bad, will repeat with a new tube of taq polymerase *I double checked the condition for PCR and there are no obvious error. *Will repeat experiment with Invitrogen supplied primers, new tube of NEX Taq2X mastermix with the control DNA ==Troubleshooting== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' |- | Control DNA Template (100ng)||1||Control DNA Template (100ng)||1 |- | Invitrogen:M13-R||2||M13 F+R (2.5uM each)||8 |- | Invitrogen:M13-F||2||Sterile Water||16 |- | Sterile Water||20||NEB Taq2X MM||25 |- | NEB Taq2X MM||25||Total||50 |- | Total||50|||| |} 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever [[File:ZhangLab_2 2011-08-24 16hr 48min.jpg|300px]] *Appears that the polymerase is not the problem, but the primers. *Check the primers ordered *Sequence ordered **M13 Reverse **CAGGAAACAGCTATGAC **Invitrogen M13R **CAGGAAACAGCTATGAC **M13 Forward **GACCGGCAGCAAAATG **Invitrogen M13F **GTAAAACGACGGCCAG *Order for M13F was reversed, will discard the forward primer and use the ones from the kit for now. *In order to avoid having to many transformations and well-spaced colonies on the LB plates in the future I should spread 10 to 100ul from each transformation vial on LB plates, for plating smaller volumes, add 20ul soc to ensure even spreading. *It will be best to measure the concentration of the purified PCR product and purified ligation product *Make sure to calculate the amount of pcr product to use with this equation x ng insert =(10*Y bp pcr product*25ng linearized pcr blunt)/3500bp pcr blunt
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