Alice:LabNotes/2008-9-3

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Experiment: Extending the cosmic probe length[edit]

previous steps and the over-view of the experiment can be found through this link [[1]]

T7 exonuclease digestion[edit]

obtained 48 reactions tubes from Ida on cpg30K V2.2
1. The tubes were purified using ethanol purification, and diluted in 100ul H2O for every 24 reactions. 
2. Add ratio of 0.5ul/tube amount of T7, which is 12ul of T7 and 11ul of NEBuffer #4 to each tube(24 reactions).
3. 25C for 2 hrs.
4. Qiaquick column purification, elute in 30ul of H2O.
5. Measure with Nanodrop.

Nanodrop result: 125.8ng/ul

TBE gel of the product after PCR and T7 digestion:
File:9-4-08.jpg

circularization reaction[edit]

the concentration of the template from last step is 125.8ng/ul, so it is equivalent to 125.8/144/330=2.64uM
reaction system 1: 1:1 ratio of template:circleHelper
reaction system 2: 1:3 ratio of template:circleHelper

reaction system 1:
template (264nM)                     2ul       
10x Ampligase Buffer                 2ul  
CircleHelper V4.2(1uM)             0.5ul
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               14ul

reaction system 2:
template (264nM)                     2ul       
10x Ampligase Buffer                 2ul  
CircleHelper V4.2(1uM)             1.5ul               
dNTP(10mM)                         0.5ul                    
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               13ul

94C 2 min -> 55C 20 min
TBU gel after the circularization reaction:
File:9-5-08.jpg

add 1.5ul Exo I &III + 10ul of product from reaction system 1
37C 2hr -> 95 10min -> 4C
Purify using Minelute columns
9/9/08: add 2.5ul Exo I&III + 20ul of product from reaction system 2
        37C 2hr -> 95 10min -> 4C
        Purify using Minelute columns

PCR (9/9/08)[edit]

H2O                                                40ul       
SYBR  green (50x)                                 0.4ul          
Forward primer AmpRV6.2_2(100uM)                  0.2ul          
Reverse primer AmpFV6.2_2(100uM)                  0.2ul
Taq master mix(2x)                                 50ul         
probe template (product after Exo I&III)           10ul

94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold
File:9-9-08 PCR after Exo I&III.jpg    TBU gel of each step:File:9-9-08 after pcr.jpg

T7 exonuclease digestion (9/9/08)[edit]

Add 2ul of T7 exonuclease to the PCR product
25C 2hr -> 4C
Purify with Qiaquick columns, and measure the concentration using Nanodrop to determine the amount of spacer needed.
Nanodrop result: 27.4ng/ul * 30ul

Insert Spacer into Circularized DNA (9/10/08)[edit]

27.4ng / (160*330) = 510nM, 5ul of template makes it 2.55 umol.

107bp spacer concentration is 156.7 ng/ul, which is 4.4 uM. We need to achieve 1:1 probe/spacer ratio, so the
volume of spacer needed is 0.6 ul. 
template                             5ul       
10x Ampligase Buffer                 2ul  
107bp Spacer                       0.6ul
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               11ul

(94C 2min -> 60C 20min ) x4 cycles

add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C
TBU gel of the results:
File:9-11-08 after spacer insertion.jpg

PCR amplification after spacer insertion[edit]

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 -> ) x35 cycles -> 72C 5min -> 15C hold

                                                   TBE gel after PCR:
File:9-11-08 PCR after spacer insertion.jpg   File:ZhangLab 2 2008-09-12 10hr 37min.jpg