Daniel:Notebook/ComboLock/2017-4-29: Difference between revisions
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>Djacobse (Created page with "=Production Run (Started Yesterday)= Back to Calendar ==Protocol-Part 4== <ol start="7"> <li>Exonuclea...") |
>Djacobse |
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=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4- | =Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-28|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Line 8: | Line 8: | ||
<li>Exonuclease Digestion</li> | <li>Exonuclease Digestion</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)</li> | ||
<li>Add 2 uL to each sample, mix by swirling pipette tip</li> | <li>Add 2 uL to each sample, mix by swirling pipette tip</li> | ||
<li>Incubate for | <li>Incubate for 2 hours at 37C</li> | ||
<li>Heat kill by incubating for 5 min at 95C</li> | <li>Heat kill by incubating for 5 min at 95C</li> | ||
</ol> | </ol> | ||
Line 37: | Line 37: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | p12RC*** (10 uM) | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2.04 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 1.24 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | RCA Primer LLRC*** (10 uM) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2.04 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 1.24 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | dNTP (10 mM) | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5.1 | |||
| align="center" align="center" valign="bottom" | 3.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | | height="15" valign="bottom" | 10X Buffer | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
Line 54: | Line 60: | ||
| align="center" align="center" valign="bottom" | 12.4 | | align="center" align="center" valign="bottom" | 12.4 | ||
|- style=" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Phi29 | | height="15" valign="bottom" | Phi29 | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0 | ||
|- style="font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | BSA (10 mg/mL) | | height="15" valign="bottom" | BSA (10 mg/mL) | ||
| align="center" align="center" valign="bottom" | 0.4 | | align="center" align="center" valign="bottom" | 0.4 | ||
Line 66: | Line 72: | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style=" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.7 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 119.34 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 75.02 | ||
|- style="font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Total | | 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" | 20 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 153 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 93 | ||
|} | |} | ||
<li>Add 15 uL master mix to each tube</li> | <li>Add 15 uL master mix to each tube</li> | ||
<li>Add 4 uL sample to appropriate tubes</li> | |||
<li>Add | |||
<li>Heat reactions up to 95C for 5 min</li> | <li>Heat reactions up to 95C for 5 min</li> | ||
<li>Cool to 55C and incubate for 15 minutes</li> | <li>Cool to 55C and incubate for 15 minutes</li> | ||
<li>Cool to 30C and add enzyme</li> | <li>Cool to 30C and add 1 uL enzyme</li> | ||
<li>Incubate for 3 hours at 30C</li> | <li>Incubate for 3 hours at 30C</li> | ||
<li>Incubate for 65 minutes at 20C to denature</li> | <li>Incubate for 65 minutes at 20C to denature</li> | ||
</ol> | |||
<li> | <li>Bglii digestion</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make | <li>Make the following reactions; '''Do not add Bglii yet'''</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
|style="background-color:#8DB4E2" width="145" height="30" | Reagent | |||
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol | |||
|style="background-color:#95B3D7" width="85" | Master Mix (16.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RCA Rxn | |||
| align="center" align="center" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer 3.1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 32.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Bglii | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 162 | |||
|- 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" | 194.4 | |||
|} | |||
<li>Aliquot 12 uL master mix into new tube</li> | |||
<li>Add 7 uL appropriate sample</li> | |||
<li>Mix with vortexer, spin down, and add 1 uL Bglii</li> | |||
<li>Incubate 2 hr 37C</li> | |||
<li>Heat kill with 65C for 20 min</li> | |||
<li>Hold at 12C</ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-5-1|Monday]] | |||
[[Category:ComboLock]] [[Category: | [[Category:ComboLock]] [[Category:20170428]] |
Latest revision as of 18:54, 1 May 2017
Production Run (Started Yesterday)[edit]
Protocol-Part 4[edit]
- Exonuclease Digestion
- Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
Reagent | Single Rxn (uL) | Master Mix X (10.2X) | Master Mix Y (6.2X) |
Enzyme | NA | NEB | Thermo |
Sample | 4 | 0 | 0 |
p12RC*** (10 uM) | 0.2 | 2.04 | 1.24 |
RCA Primer LLRC*** (10 uM) | 0.2 | 2.04 | 1.24 |
dNTP (10 mM) | 0.5 | 5.1 | 3.1 |
10X Buffer | 2 | 20.4 | 12.4 |
Phi29 | 1 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 4.08 | 0 |
nfH2O | 11.7 | 119.34 | 75.02 |
Total | 20 | 153 | 93 |
Reagent | Single Rxn Vol | Master Mix (16.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 32.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 162 |
Total | 20 | 194.4 |
Continued Monday