Daniel:Notebook/ComboLock/2017-1-26: Difference between revisions

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<ol type="a">
<ol type="a">
<li>109.2 uL nfH2o</li>
<li>109.2 uL nfH2o</li>
<li>5.2 uL AmpF</li</li>
<li>5.2 uL AmpF</li>
<li>130 uL 2X Kapa SYBR Fast Master Mix</li>
<li>130 uL 2X Kapa SYBR Fast Master Mix</li>
<li>Add 47 uL master mix to each well</li>
<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</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) 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="100" height="45" | Sample
| width="100" | Condition
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Sample Vol (uL)
| width="85" | 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 6AC
| align="center" | Epicentre
| align="center" | A1
| 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 6BC
| align="center" | Epicentre
| align="center" | A2
| 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 6AD
| align="center" | Epicentre 1:10
| align="center" | A3
| 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"  valign="bottom" | Sample 6BD
| align="center" | Epicentre 1:10
| align="center" | A4
| 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" | &nbsp;
| align="center" valign="bottom" | A5
| align="center" align="center" valign="bottom" | 24
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 21
| align="center" align="center" valign="bottom" | 50
|}


<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
Line 39: Line 114:
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li>
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li>
<li>Rinse gel and image in gel doc</li>
<li>Rinse gel and image in gel doc</li>
</ol></ol>
==Library Prep==
Those bands are interesting to me. Time to sequence.
'''Sample Matrix'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center"
| width="160" height="30" | Sample
| width="85" | Sample Amt
| width="85" | Pre/Post RCA
| width="85" | AmpR Index
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 6C (AB)
| align="center" valign="bottom" | 1 amol
| align="center" valign="bottom" | Epi
| align="center" align="center" valign="bottom" | 22
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 6D (AB)
| align="center" valign="bottom" | 1 amol
| align="center" valign="bottom" | Epi 1:10
| align="center" align="center" valign="bottom" | 23
|}
<ol>
<li>Size Select Gel</li>
<ol type="A">
<li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li>
<li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li>
<li>Aliquot 48/24 uL per sample/ladder lane into each well</li>
<li>Run gel for 23 minutes at 230V</li>
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li>
<li>Rinse gel and image in gel doc</li>
<li>Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li>
<li>Image in gel doc</li>
<gallery perrow=2 heights=300px widths=300px>
File:2017-01-26-RCATest-Epicentre-SizeSelect.png|Before Image
File:2017-01-26-RCATest-Epicentre-SizeSelect-After.png|After Image
</gallery>
<li>Centrifuge at 12000 rpm for 1.5 minutes</li>
<li>Add 500 uL TE to each sample</li>
<li>Incubate 4 hours at 37C with vigorous shaking;</li>
<li>Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom</li>
<li>Extract supernatant and add to nanosep column</li>
<li>Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant</li></ol>
<li>Ethanol Precipitation</li>
<ol type="A">
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li>
<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1-27|tomorrow]]</li>
</ol></ol>
</ol></ol>


Line 47: Line 176:
File:2017-01-26-RCATest-Epicentre.png|Gel image
File:2017-01-26-RCATest-Epicentre.png|Gel image
</gallery>
</gallery>
==Library Prep (Started [[Daniel:Notebook/ComboLock/2017-1-25|Yesterday]])==
<ol start="2">
<li>Ethanol Precipitation</li>
<ol type="A" start="3">
<li>Centrifuge for 30 minutes at 12500 rpm at 4C</li>
<li>Remove supernatant and add 750 uL chilled 70% EtOH</li>
<li>Centrifuge for 15 minutes at 12500 rpm at 4C</li>
<li>Remove supernatant; dry in vacuum centrifuge for 15 minutes</li>
<li>Resuspend in 20 uL H2O and consolidate samples</li>
<li>Measure with Qbit</li>
</ol></ol>
[[Category:ComboLock]] [[Category:20170123]]

Latest revision as of 23:22, 30 January 2017

RCA Test (Started Monday)[edit]

Back to Calendar

Epicentre Test (Started Yesterday)[edit]

Sample Matrix

  1. qPCR
    1. Make 5.2X qPCR master mix according to following recipe
      1. 109.2 uL nfH2o
      2. 5.2 uL AmpF
      3. 130 uL 2X Kapa SYBR Fast Master Mix
      4. Add 47 uL master mix to each well
      5. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
      6. 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 6AC Epicentre A1 22 2 25 1 1 21 50
        Sample 6BC Epicentre A2 22 2 25 1 1 21 50
        Sample 6AD Epicentre 1:10 A3 23 2 25 1 1 21 50
        Sample 6BD Epicentre 1:10 A4 23 2 25 1 1 21 50
        NTC   A5 24 2 25 1 1 21 50
      7. 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
    2. TBE Gel
      1. Mix 64 uL TBE, 16 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 23 minutes at 230V
      6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
      7. Rinse gel and image in gel doc

    Library Prep[edit]

    Those bands are interesting to me. Time to sequence.

    Sample Matrix

    Sample Sample Amt Pre/Post RCA AmpR Index
    Sample 6C (AB) 1 amol Epi 22
    Sample 6D (AB) 1 amol Epi 1:10 23
    1. Size Select Gel
      1. Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
      2. Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
      3. Aliquot 48/24 uL per sample/ladder lane into each well
      4. Run gel for 23 minutes at 230V
      5. Open gel and stain with 3 uL SYBR Gold for 3 minutes
      6. Rinse gel and image in gel doc
      7. Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
      8. Image in gel doc
      9. Centrifuge at 12000 rpm for 1.5 minutes
      10. Add 500 uL TE to each sample
      11. Incubate 4 hours at 37C with vigorous shaking;
      12. Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
      13. Extract supernatant and add to nanosep column
      14. Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
    2. Ethanol Precipitation
      1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
      2. Incubate at -80C overnight; continued tomorrow

    Results[edit]

    Library Prep (Started Yesterday)[edit]

    1. Ethanol Precipitation
      1. Centrifuge for 30 minutes at 12500 rpm at 4C
      2. Remove supernatant and add 750 uL chilled 70% EtOH
      3. Centrifuge for 15 minutes at 12500 rpm at 4C
      4. Remove supernatant; dry in vacuum centrifuge for 15 minutes
      5. Resuspend in 20 uL H2O and consolidate samples
      6. Measure with Qbit