Daniel:Notebook/ComboLock/2017-1-7: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Version 2 Oligos (Started Yesterday)= Back to Calendar ==Protocol== <ol start="8"> <li>Exonuclease Dig...")
 
>Djacobse
 
(10 intermediate revisions by the same user not shown)
Line 3: Line 3:
[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]


==Protocol==
==Protocol Part 3==


<ol start="8">
<ol start="8">
<li>Exonuclease Digestion</li>
<li>Exonuclease Digestion</li>
<ol type="A">
<ol type="A">
<li>Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)</li>
<li>Mix 15 uL Exo I (20U/uL) and 3 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 1 hour at 37C</li>
<li>Incubate for 1 hour at 37C</li>
Line 18: Line 18:
<li>Add 15 uL master mix to each tube</li>
<li>Add 15 uL master mix to each tube</li>
<li>Add sample to new reaction tubes according to following table</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="170" height="30" | Reagent
| width="65" | uL Added
| width="65" | 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 (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 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 3 hours</li>
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol>
==Protocol Part 4==
<li>qPCR</li>
<ol type="A">
<li>Make 9.2X qPCR master mix according to following recipe</li>
<ol type="a">
<li>193.2 uL nfH2O</li>
<li>230 uL SYBR Fast</li>
<li>9.2 uL 10 uM AmpF</li>
</ol>
<li>Add 47 uL master mix to each well</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section</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="140" height="45" | Sample
| width="160" | Condition
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Sample Vol (uL)
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 10 uM Forward Primer
| width="65" | 10 uM Reverse Primer
| width="65" | H2O
| width="65" | Total Volume (uL)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1A
| align="center" | Sample
| align="center" | A1
| align="center" align="center" | 20
| 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
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1B
| align="center" | Sample
| align="center" | A2
| align="center" align="center" | 20
| 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
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 2A
| align="center" | No Template
| align="center" | A3
| 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
|- 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" 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
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3A
| align="center" | No C Probe
| align="center" | A5
| 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
|- 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" 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
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4A
| align="center" | No Lock Phosphate
| align="center" | A7
| 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
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4B
| align="center" | No Lock Phosphate
| align="center" | A8
| 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
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" valign="bottom" | qPCR Primers Only
| align="center" | H1
| 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" | 23
| align="center" align="center" | 50
|}
<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:20170107-qPCR-v2Oligos.png|qPCR Curve
File:2017-01-09-v2OligoSet.png|Gel Image
</gallery>
[[Category:ComboLock]] [[Category:20170106]]

Latest revision as of 20:10, 9 January 2017

Version 2 Oligos (Started Yesterday)[edit]

Back to Calendar

Protocol Part 3[edit]

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

    Protocol Part 4[edit]

  3. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample A1 20 2 25 1 1 21 50
      Sample 1B Sample A2 20 2 25 1 1 21 50
      Sample 2A No Template A3 21 2 25 1 1 21 50
      Sample 2B No Template A4 21 2 25 1 1 21 50
      Sample 3A No C Probe A5 22 2 25 1 1 21 50
      Sample 3B No C Probe A6 22 2 25 1 1 21 50
      Sample 4A No Lock Phosphate A7 23 2 25 1 1 21 50
      Sample 4B No Lock Phosphate A8 23 2 25 1 1 21 50
      NTC qPCR Primers Only H1 24 2 25 1 1 23 50
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 80 uL TBE, 20 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]