Alice:LabNotes/2008-10-28

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Revision as of 04:40, 29 October 2008 by >Zsakura2 (→‎=spacer insertion)
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=spacer insertion

For the product and procedure from previous steps, please follow this link (10/14/08 LabNote): [[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 Repeat 2: PCR after circlehelper insertion(10/21/08) from link above) was purified
with Qiaquick columns,elute DNA in 30ul ddH2O. 
Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, 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. Dilute the spacer to 100nM since the PCR product concentration
is quite low (we need 1:1 ratio of primer:template)
Prepare the oligo: Add 10ul of each primer to 990ul of ddH2O to dilute them to 100nM
                   Denature at 95C for 5min, and then anneal at 65C for 20min.
Prepare the probe: take of template (1) and 


                   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