Alice:LabNotes/2008-10-3

From ZhangLabWiki
Jump to navigation Jump to search

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