Alice:LabNotes/2008-10-3
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Experiment: Elongating Cosmic Probe length[edit]
template used is the probe after circleHelper insertion (see 9-15-08 labnote), which is 160bp long. The nanodrop result is 31.8 ng/ul, which is 31.8 ng/(160x330) = 602.3 nM.
The amount of insertion spacer oligo ordered was 2nmol, and the reverse arm was 4.8nmol. Diluting them using 200ul and 480ul of ddH2O respectively yields final concentration of 10uM.
When using 10ul of template, we obtain 10ul x 0.6023uM = 6.026umol. so the amount of both spacer oligo needed is 6.026umol/10uM= 0.6ul.
Prepare the oligo: combining 0.6ul of reverse and insertion oligo, which is a total of 1.2 ul. denature at 95C for 5min, and then anneal at 65C for 20min.
Prepare the probe: take out 10ul of template (product after circlehelper insertion and purification) 95C for 5 min, then 65C for 1 min, add 1ul of 10x ampligase buffer, 0.5ul of ampligase, 1.2 of primer anneal at 65C for 30 min. add 1.5ul Exo I&III 37C 2hr -> 95C 5min -> 4C
PCR amplification after spacer insertion: H2O 70.6ul 10x Stoffel buffer 10ul MgCl2(25mM) 6ul dNTP(10mM each, NEB) 2ul Primer mix 100uM (Ap1V4IU +Ap2V4) 0.4ul 50x SYBG I 0.4ul template(from last step) 10ul Jumpstart Taq 0.6ul 94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x25 cycles -> 72C 5min -> 15C hold TBE gel on PCR result: File:10-6-08 PCR after spacer insertion.jpg File:ZhangLab 2 2008-10-06 14hr 52min.jpg