Daniel:Notebook/ComboLock/2017-1-25: Difference between revisions

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>Djacobse
(Created page with "==Protocol== <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>17.2 uL AmpF-CLv3</li> <li>17.2...")
 
>Djacobse
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=Circularization Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Monday]])=
[[Daniel:Notebook/ComboLock|Back to Calendar]]
==Protocol==
==Protocol==


<ol>
<li>qPCR</li>
<li>qPCR</li>
<ol type="A">
<ol type="A">
Line 11: Line 16:
<li>Add 48 uL master mix to each well</li>
<li>Add 48 uL master mix to each well</li>
<li>Add 2 uL sample according to table</li>
<li>Add 2 uL sample 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="85" | Product Amt
| width="85" | Pre/Post RCA
| width="65" | Lane
| 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 0A
| align="center" | 1pmol
| align="center" | Pre
| align="center" | A1
| 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 0B
| align="center" | 1pmol
| align="center" | Pre
| align="center" | A2
| 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" | 10 fmol
| align="center" | Pre
| align="center" | A3
| 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" | 10 fmol
| align="center" | Pre
| align="center" | A4
| 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" | 100 amol
| align="center" | Pre
| align="center" | A5
| 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" | 100 amol
| align="center" | Pre
| align="center" | A6
| 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 6A
| align="center" | 1 amol
| align="center" | Pre
| align="center" | A7
| 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 6B
| align="center" | 1 amol
| align="center" | Pre
| align="center" | A8
| 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 0AX
| align="center" | 1pmol
| align="center" | Post
| align="center" | H1
| 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:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 0BX
| align="center" | 1pmol
| align="center" | Post
| align="center" | H2
| 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 2AX
| align="center" | 10 fmol
| align="center" | Post
| align="center" | H3
| 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:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2BX
| align="center" valign="bottom" | 10 fmol
| align="center" | Post
| align="center" | H4
| 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 4AX
| align="center" | 100 amol
| align="center" | Post
| align="center" | H5
| 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:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4BX
| align="center" | 100 amol
| align="center" | Post
| align="center" | H6
| 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 6AX
| align="center" | 1 amol
| align="center" | Post
| align="center" | H7
| 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:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 6BX
| align="center" | 1 amol
| align="center" | Post
| align="center" | H8
| 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
|}
<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
<ol type="a">
<ol type="a">

Revision as of 16:42, 25 January 2017

Circularization Test (Started Monday)

Back to Calendar

Protocol

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF-CLv3
      3. 17.2 uL AmpR-CLv3
      4. 430 uL Kapa SYBR Fast
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to table
    4. Sample Product Amt Pre/Post RCA Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 0A 1pmol Pre A1 2 25 1 1 21 50
      Sample 0B 1pmol Pre A2 2 25 1 1 21 50
      Sample 2A 10 fmol Pre A3 2 25 1 1 21 50
      Sample 2B 10 fmol Pre A4 2 25 1 1 21 50
      Sample 4A 100 amol Pre A5 2 25 1 1 21 50
      Sample 4B 100 amol Pre A6 2 25 1 1 21 50
      Sample 6A 1 amol Pre A7 2 25 1 1 21 50
      Sample 6B 1 amol Pre A8 2 25 1 1 21 50
      Sample 0AX 1pmol Post H1 2 25 1 1 21 50
      Sample 0BX 1pmol Post H2 2 25 1 1 21 50
      Sample 2AX 10 fmol Post H3 2 25 1 1 21 50
      Sample 2BX 10 fmol Post H4 2 25 1 1 21 50
      Sample 4AX 100 amol Post H5 2 25 1 1 21 50
      Sample 4BX 100 amol Post H6 2 25 1 1 21 50
      Sample 6AX 1 amol Post H7 2 25 1 1 21 50
      Sample 6BX 1 amol Post H8 2 25 1 1 21 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
  2. 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