Editing
Daniel:Notebook/ComboLock/2017-1-19
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=Version 3 Oligos (Started [[Daniel:Notebook/ComboLock/2017-1-13|Thursday, Jan 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==RCA Repeat (Started [[Daniel:Notebook/ComboLock/2017-1-18|Yesterday]])== <ol start="2"> <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="100" height="30" | Master Mix | width="120" | Primer Pair | width="87" | Forward Primer (10 uM) uL | width="89" | Reverse Primer (10 uM) uL | width="65" | uL nfH2O | width="106" | uL Kapa SYBR Fast Master Mix 2X |- style="font-size:12pt" | height="15" valign="bottom" | MM1 | align="center" | AmpF/AmpR | align="center" align="center" | 9.2 | align="center" | NA | align="center" align="center" | 193.2 | align="center" align="center" | 230 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | MM2 | align="center" | Primer12RC/AmpRInd25 | align="center" align="center" | 9.2 | align="center" align="center" | 9.2 | align="center" align="center" | 193.2 | align="center" align="center" | 230 |} <li>Add 47 (MM1) or 48 (MM2) uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (10 uM, MM1 only) according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="105" height="45" | Sample | width="140" | Condition | width="65" | Lane | width="106" | Primer Pair | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="61" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM Forward Primer | width="65" | 10 uM Reverse Primer | width="65" | H2O | width="71" | Total Volume (uL) | width="65" | Master Mix |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | A1 | align="center" | AmpF/AmpR | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | A2 | align="center" | AmpF/AmpR | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | A3 | align="center" | AmpF/AmpR | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" | A4 | align="center" | AmpF/AmpR | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No C Probe | align="center" | A5 | align="center" | AmpF/AmpR | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No C Probe | align="center" | A6 | align="center" | AmpF/AmpR | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 4A | align="center" | No PCCB Phosphate | align="center" | A7 | align="center" | AmpF/AmpR | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="30" | Sample 4B | align="center" | No PCCB Phosphate | align="center" | A8 | align="center" | AmpF/AmpR | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample-PostRCA | align="center" | H1 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample-PostRCA | align="center" | H2 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 4A | align="center" | No PCCB Phosphate | align="center" | H3 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="30" | Sample 4B | align="center" | No PCCB Phosphate | align="center" | H4 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample-PreRCA | align="center" | H5 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample-PreRCA | align="center" | H6 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" | Sample 4A | align="center" | No PCCB PO4-PreRCA | align="center" | H7 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="background-color:#DA9694;font-size:12pt" |style="font-weight:bold" height="30" | Sample 4B | align="center" | No PCCB PO4-PreRCA | align="center" | H8 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 | align="center" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" | D1 | align="center" | AmpF/AmpR | align="center" align="center" | 26 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 | align="center" | MM1 |- style="background-color:#DA9694;font-size:12pt" | height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" | D2 | align="center" | Primer12RC/AmpR | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 | align="center" | MM2 |} <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 80 uL TBE, 20 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170119-qPCR-v3Oligos-AmpFAmpR.png|qPCR Curve-AmpF and AmpR File:20170119-qPCR-v3Oligos-Primer12RCAmpR.png|qPCR Curve-Primer12RC and AmpR File:2017-01-19-v3Oligos-RCA-2.png|Gel Image-AmpF and AmpR File:2017-01-19-v3Oligos-NoAmpF.png|Gel Image-Primer12RC and AmpR </gallery> ===Discussion=== Ummmm. So the AmpR/Primer12RC should only amplify circular product (yesterday's p12/p24 combo is good for circular product and bridge). So once again it showed up in both pre and post RCA reactions, but the normal AmpR/AmpF pairing (necessary for sequencing) showed nothing. Also, assuming I didn't mix up the samples, it kinda seems like there is more product Pre-RCA than post. Going off the qPCR curves (generally a bad idea but probably comparable between these two) it looks like the "PreRCA" sample fraction is ~4X more than the "PostRCA" fraction. This actually makes a ton of sense if there was no amplification from RCA. Both PCRs had 2 uL sample added to them, but the "PostRCA" fraction got 5 uL (out of 20) of "PreRCA" fraction as input. So basically I put the "PreRCA" fraction into water, or might as well have. ==RCA Test== Since it seems like its RCA that is the problem I will do RCA again, this time testing both AmpF and AmpR as (dirty) primers. To save on sample space, I'll use 1 and 4 again (4 is a suitable negative control). I should know whether or not the product amplifies in even half an hour, so we're gonna go with that (measure using Qubit) <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.5X Master mix according to table below</li> <li>Add 15 uL master mix to each tube</li> <li>Add sample to new reaction tubes according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="160" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (8.5X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (AmpF or AmpRInd21; 10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 21.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 6.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Phi29 Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 73.95 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 127.5 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> <li>Qubit Measure</li> <ol type="A"> <li>Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)</li> <li>Add 190 uL dye/buffer and 10 uL standard to standard tubes</li> <li>Add 199 uL dye/buffer and 1 uL sample to sample tubes</li> <li>Measure in Qubit</li> </ol></ol> ===Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="91" height="24" | Sample Name | width="60" | 1A | width="76" | 1B | width="71" | 1AY | width="65" | 1BY | width="65" | 1AF | width="65" | 1BF | width="65" | 4AF | width="65" | 4BF | width="65" | 1AR | width="65" | 1BR | width="65" | 4AR | width="65" | 4BR |- style="font-size:12pt" | height="45" | Sample Condition | align="center" | Sample-PreRCA | align="center" | Sample-PreRCA | align="center" | Sample | align="center" | Sample | align="center" | Sample | align="center" | Sample | align="center" | No Phosphate (PCCB) | align="center" | No Phosphate (PCCB) | align="center" | Sample | align="center" | Sample | align="center" | No Phosphate (PCCB) | align="center" | No Phosphate (PCCB) |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | RCA Primer | align="center" | None | align="center" | None | align="center" | LLRC (Mine) | align="center" | LLRC (Mine) | align="center" | AmpF | align="center" | AmpF | align="center" | AmpF | align="center" | AmpF | align="center" | AmpR | align="center" | AmpR | align="center" | AmpR | align="center" | AmpR |- style="font-size:12pt" | height="15" | Conc (ng/uL) | align="center" align="center" | 0.81 | align="center" align="center" | 1.27 | align="center" align="center" | 0.72 | align="center" align="center" | 0.62 | align="center" align="center" | 35.1 | align="center" align="center" | 46.9 | align="center" align="center" | 35 | align="center" align="center" | 40.7 | align="center" align="center" | 25.3 | align="center" align="center" | 26.8 | align="center" align="center" | 31.5 | align="center" align="center" | 30.2 |} ===Discussion (RCA Test)=== From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF '''and''' AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers. Continued [[Daniel:Notebook/ComboLock/2017-1-20|tomorrow]] [[Category:ComboLock]] [[Category:20170113]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information