Alice:LabNotes/2008-9-15: Difference between revisions

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<font size=10><b>Experiment: extending the probe length </b> </font>
<font size=4><b>Experiment: extending the probe length </b> </font>
  After PCR amplification after CircleHelper insertion, T7 exonuclease digestion was performed. And the detailed procedures can  
  After PCR amplification after CircleHelper insertion, T7 exonuclease digestion was performed. And the detailed procedures can  
  be found through this link: [[https://genome-tech.ucsd.edu/LabNotes/index.php?title=Alice:LabNotes/2008-9-3]]
  be found through this link: [[https://genome-tech.ucsd.edu/LabNotes/index.php?title=Alice:LabNotes/2008-9-3]]
Line 32: Line 32:
  5. Let dry in hood ~ 10 min.
  5. Let dry in hood ~ 10 min.
  6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
  6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Nanodrop result: 31.8ng/ul


==Insert Spacer into Circularized DNA (9/16/08)==
==Insert Spacer into Circularized DNA (9/16/08)==
Line 48: Line 50:
  (94C 2min -> 60C 20min ) x4 cycles
  (94C 2min -> 60C 20min ) x4 cycles
   
   
  [[Image:ZhangLab_2 2008-09-16 10hr 38min-1.jpg]]  
  [[Image:ZhangLab_2 2008-09-16 10hr 38min-1.jpg]] correction: the middle lane is the product after spacer insertion, not PCR amplification
   
   
  add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C
  add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C
Line 63: Line 65:
  94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x25 cycles -> 72C 5min -> 15C hold
  94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x25 cycles -> 72C 5min -> 15C hold


  TBU gel on PCR result (9/17/08)
  TBU gel on PCR result
  [[Image:ZhangLab_2 2008-09-17 12hr 32min.jpg]]
  [[Image:ZhangLab_2 2008-09-17 12hr 32min.jpg]]
Result: Again, the gel showed no distinct band at size 250bp, which is the result we are interested in. Therefore a gel cut
will be performed on 107bp spacer and reinsert into the probe.
Repeat the steps Page Denaturing Gel, Purify the product from the gel, and precipitation from the above protocol.
==Page Denaturing Gel of the spacer (9/17/08)==
Image of the 2D TBU gel:
[[Image:ZhangLab_2 2008-09-17 16hr 00min.jpg]]
Purify gel and precipitate
==Insert Spacer into Circularized DNA (9/18/08)==
template                            5ul     
10x Ampligase Buffer                2ul 
107bp Spacer                      0.4ul
dNTP(10mM)                        0.5ul
Stoffel fragment(10U/ul)          0.5ul
Ampligase(5U/ul)                  0.5ul
ddH2O                              11ul
(94C 2min -> 60C 20min ) x4 cycles
[[Image:ZhangLab_2 2008-09-18 13hr 16min.jpg]] 
add 2ul Exo I/III-> 37C 2h -> 94C 5mins >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
TBU gel on PCR result

Latest revision as of 20:09, 3 October 2008

Experiment: extending the probe length

After PCR amplification after CircleHelper insertion, T7 exonuclease digestion was performed. And the detailed procedures can 
be found through this link: [[1]]

Page Denaturing Gel[edit]

1. Add TBE-Urea sample buffer (2x) in each tube.
2. Mix 0.5 uL Invitrogen 10bp ladder with 3.5 uL dH2O, 4 uL 2x TBE-Urea sample buffer.
3. Denature at 75C for 8 min, then quickly transfer to ice, wait for 1 min.
4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea. 
5. Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min.
6. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min.

2D gel to take out the band we are interestd in (160bp):
File:ZhangLab 2 2008-09-15 12hr 53min.jpg

Purify the product from the gel[edit]

1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use
G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. 
2.Cut out the correct band, split into two half and transfer each half to one shearing assembly.
3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
4. Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C
incubator, shake the tubes at low speed for 45 min.
5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer
supernatant to 1.5mL tube.

Precipitation[edit]

1. Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol.
2. Add 1uL Glycoblue so that one can precipitate better and see the DNA.
3. Freeze in -70C for 20 min, spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette.
4. Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with 
first large tip and then small tip to remove as much liquid as possible.
5. Let dry in hood ~ 10 min.
6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Nanodrop result: 31.8ng/ul

Insert Spacer into Circularized DNA (9/16/08)[edit]

19.8ng / (160*330) = 375nM, 5ul of template makes it 1.88 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 0.4 ul. 
template                             5ul       
10x Ampligase Buffer                 2ul  
107bp Spacer                       0.4ul
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               11ul

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

File:ZhangLab 2 2008-09-16 10hr 38min-1.jpg  correction: the middle lane is the product after spacer insertion, not PCR amplification

add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C

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 -> ) x25 cycles -> 72C 5min -> 15C hold
TBU gel on PCR result
File:ZhangLab 2 2008-09-17 12hr 32min.jpg
Result: Again, the gel showed no distinct band at size 250bp, which is the result we are interested in. Therefore a gel cut
will be performed on 107bp spacer and reinsert into the probe.
Repeat the steps Page Denaturing Gel, Purify the product from the gel, and precipitation from the above protocol.

Page Denaturing Gel of the spacer (9/17/08)[edit]

Image of the 2D TBU gel:
File:ZhangLab 2 2008-09-17 16hr 00min.jpg

Purify gel and precipitate

Insert Spacer into Circularized DNA (9/18/08)[edit]

template                             5ul       
10x Ampligase Buffer                 2ul  
107bp Spacer                       0.4ul
dNTP(10mM)                         0.5ul
Stoffel fragment(10U/ul)           0.5ul
Ampligase(5U/ul)                   0.5ul
ddH2O                               11ul

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

File:ZhangLab 2 2008-09-18 13hr 16min.jpg   
add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C

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 -> ) x25 cycles -> 72C 5min -> 15C hold
TBU gel on PCR result