Alice:LabNotes/2008-10-28
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=spacer insertion
For the product and procedure from previous steps, please follow this link (10/10/08 lab note): [[1]]
template used: (1) PCR product of repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III (2) PCR product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III
PCR results from lane 1 and 3 (see section above) was purified with Qiaquick columns,elute DNA in 30ul ddH2O. Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, and 2ul of Lambda Exo. 37C 2hrs -> 90C 10 min Purify it using Qiaquick columns again and use Nanodrop to test the concentration. Nanodrop result: (1) 5.7 ng/ul = 119nM (2) 10.1 ng/ul = 680 nM Note: this proves that treating the PCR product with exonuclease gives about twice as much %yield as comparing to T7 exonuclease.
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.
Prepare the oligo: denature at 95C for 5min, and then anneal at 65C for 20min.
Prepare the probe:
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