Alice:LabNotes/2008-9-15: Difference between revisions
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< | <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]] | ||
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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)== | ||
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(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 64: | 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 | 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