Alice:LabNotes/2008-10-6

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Experiment: Extending Cosmic Probe Length[edit]

PCR amplification of cosmic probe[edit]

PCR reaction system                                      x48              
H2O                                       50ul          2400ul              
SYBR  green (50x)                        0.4ul          19.2ul
primer mix (100uM)                       0.2ul          9.6ul                 
Taq master mix(2x)                        50ul          2400ul 
probe template (cosmic)                 0.05ul          2.4ul
 
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min ->
15C hold

Purify the product using ethanol purification:
1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, 6 mL 100% Ethanol.
2. Store at -70C for more than 20 min.
3. Spin at 4000rpm for 15 min at 4C.
4. Remove all liqid, you should see a blue pallette at the bottom of the tube.
5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube.
6. Spin at 10000 rpm for 5 min at 4C.
7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 100 uL dH2O.

T7 exonuclease digestion (10/7/08)[edit]

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: 24.2ng/ul * 35ul

Circularization reaction[edit]

the concentration of the template from last step is 24.2 ng/ul, which is 24.2/145/330= 509nM
reaction system 1:                                  reaction system 2:           
template (509nM)                     2ul            template (509nM)                     1ul 
10x Ampligase Buffer                 2ul            10x Ampligase Buffer                 2ul
CircleHelper V4.2(1uM)               1ul            CircleHelper V4.2(1uM)               2ul 
dNTP(10mM)                         0.5ul            dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul            Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul            Ampligase(5U/ul)                   0.5ul
ddH2O                             14.5ul            ddH2O                               15ul

94C 2 min -> 55C 20 min
File:ZhangLab 2 2008-10-08 15hr 28min.jpg
result: the band at 100bp has much higher intensity than all other bands, however, it is not the band we expected. Also, few more
experiments will be done to determine the content of this 100bp band before I continue with the next step.
add 1.5ul Exo I &III + 10ul of product from reaction system 1
37C 2hr -> 95 10min -> 4C
Purify using Minelute columns

circularization under different reaction system (10/9/08)[edit]

reaction system:             1           2           3            4           5
template                     +           +           +            -           +
circleHelper                 +           +           +            +           -
Ligase                       +           -           +            +           +
Stoffel fragment             +           +           -            +           +

template (509nM)                     1ul 
10x Ampligase Buffer                 2ul
CircleHelper V4.2(1uM)               2ul 
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               15ul
94C 2 min -> 55C 20 min
File:ZhangLab 2 2008-10-09 15hr 52min.jpg
result: the primer of the first PCR step is not phosphorylated at the 5'end, therefore the ligase would not be able to work. The gel
above showed very interesting results, whereas looks like the template and the stoffel fragment are having some unspecific activities.
However, I am not entirely sure what happened within each reaction, especially with the 100bp band. The steps will be repeated using
phosphorylated primer, and the same reaction system will be used again later to compare the results.

Repeat circularization steps[edit]

PCR reaction system                                               
H2O                                        50ul                     
SYBR  green (50x)                         0.4ul         
primer AP1V4(100uM) (phosphorylated)      0.2ul 
primer AP2V4(100uM)                       0.2ul 
Taq master mix(2x)                         50ul      
probe template (cosmic)                  0.05ul         

94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 
T7 exonuclease digestion: 
Add ratio of 0.5ul/tube amount of T7
25C for 2 hrs.
(Only two out of the 10 tubes prepared is used for purification and later steps)
Qiaquick column purification, elute in 30ul of H2O.
Measure with Nanodrop. Result: 45.4ng/ul
Circularization reaction: (10/10/08)
the concentration of the template from last step is 45.4ng/ul, which is 956nM.

reaction system:             1           2           3            4           5
template                     +           +           +            -           +
circleHelper                 +           +           +            +           -
Ligase                       +           -           +            +           +
Stoffel fragment             +           +           -            +           +

template (956nM)                     1ul 
10x Ampligase Buffer                 2ul
CircleHelper V4.2(1uM)               1ul 
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               15ul
94C 2 min -> 55C 20 min

add 2ul Exo I &III to the product after Exo  I&III
37C 2hr -> 95 10min -> 4C
Purify using Minelute columns
TBU gel of the products:
File:ZhangLab 2 2008-10-10 15hr 39min-.jpg
PCR after circlehelper insertion:
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