Daniel:Notebook/ComboLock/2017-2-10: Difference between revisions

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<ol type="A">
<ol type="A">
<li>Make qPCR master mixes according to following recipes</li>
<li>Make qPCR master mixes according to following recipes</li>
'''Master Mix Recipes'''


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| 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"
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="1005" height="45" | Master Mix
| width="85" height="45" | Master Mix NoX
| width="85" | No X
| width="105" | Forward Primer Amt (10 uM)
| width="190" | Primer Pair
| width="105" | Reverse Primer Amt (10 uM)
| width="82" | Forward Primer Amt (10 uM)
| width="85" | uL nfH2O
| width="80" | Reverse Primer Amt (10 uM)
| width="105" | uL 2X Kapa SYBR Fast
| width="65" | uL nfH2O
| width="85" | Total
| width="65" | uL 2X Kapa SYBR Fast
 
| width="65" | Total
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 73
| align="center" | 73
| align="center" | 73
| align="center" | 1168
| align="center" | 1460
|style="font-weight:bold" align="center" | 2774
 
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 63
| align="center" | 63
| align="center" | 63
| align="center" | 1008
| align="center" | 1260
|style="font-weight:bold" align="center" | 2394
 
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 2.2
| align="center" | 2.2
| align="center" | 2.2
| align="center" | 35.2
| align="center" | 44
|style="font-weight:bold" align="center" | 83.6
 
|}
 
'''Plate Organization'''
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#DA9694;font-size:12pt" align="center"
| width="65" height="28" | Plate
| width="85" | Master Mix Quantity
| width="65" | Samples
| width="65" | Target
| width="150" | Primer Pair


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | MMC1
| height="15"  valign="bottom" | 1
| align="center"  valign="bottom" | 73
| align="center" valign="bottom" | Primer24/Primer4RC
| align="center" align="center" valign="bottom" | 73
| align="center" align="center" valign="bottom" | 73
| align="center" valign="bottom" | 1-5
| align="center" valign="bottom" | Template
| align="center" valign="bottom" | CLv3-AmpF/CLv3-AmpR
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 2
| align="center" align="center" valign="bottom" | 63
| align="center" valign="bottom" | 6-10
| align="center" valign="bottom" | Template
| align="center" valign="bottom" | CLv3-AmpF/CLv3-AmpR
|- style="font-size:12pt"
| height="15"  valign="bottom" | 3
| align="center" align="center" valign="bottom" | 73
| align="center" align="center" valign="bottom" | 73
| align="center" align="center" valign="bottom" | 1168
| align="center" valign="bottom" | 1-5
| align="center" align="center" valign="bottom" | 1460
| align="center" valign="bottom" | C1
|style="font-weight:bold" align="center" align="center" valign="bottom" | 2774
| align="center" valign="bottom" | primer24/primer4RC


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | MMC2
| height="15"  valign="bottom" | 4
| align="center" valign="bottom" | 2.2
| align="center" align="center" valign="bottom" | 63
| align="center" valign="bottom" | Primer6/Primer12RC
| align="center" valign="bottom" | 6-10
| align="center" align="center" valign="bottom" | 2.2
| align="center" valign="bottom" | C1
| align="center" align="center" valign="bottom" | 2.2
| align="center" valign="bottom" | primer24/primer4RC
| align="center" align="center" valign="bottom" | 35.2
| align="center" align="center" valign="bottom" | 44
|style="font-weight:bold" align="center" align="center" valign="bottom" | 83.6


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | MMT
| height="15"  valign="bottom" | 5
| align="center" valign="bottom" | 2.2
| align="center" align="center" valign="bottom" | 73
| align="center" valign="bottom" | PCCTAmpF/PCCT-AmpR
| align="center" valign="bottom" | 1-5
| align="center" align="center" valign="bottom" | 2.2
| align="center" valign="bottom" | C2
| align="center" align="center" valign="bottom" | 2.2
| align="center" valign="bottom" | primer6/primer12RC
| align="center" align="center" valign="bottom" | 35.2
 
| align="center" align="center" valign="bottom" | 44
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" align="center" align="center" valign="bottom" | 83.6
| height="15" valign="bottom" | 6
| align="center" align="center" valign="bottom" | 63
| align="center" valign="bottom" | 6-10
| align="center" valign="bottom" | C2
| align="center" valign="bottom" | primer6/primer12RC


|}
|}


<li>Add 48 uL master mix to each well</li>
<li>Add 38 uL master mix to each well</li>
<li>Add 2 uL sample according to table in following section</li>
<li>Add 2 uL sample according to table in following section</li>
<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
Line 62: Line 110:
<li>72C 15 sec</li>
<li>72C 15 sec</li>
<li>plate read</li>
<li>plate read</li>
<li>goto b x24</li>
<li>goto b x40</li>
<li>72C 2 min</li>
<li>72C 2 min</li>
<li>16C hold</li>
<li>16C hold</li>
</ol></ol>
</ol></ol></ol>


==Plate Layouts==
==Plate Layouts==
'''A Plate (Samples 1-5)'''


[[Image:20170209-Plate1-5.png|600px]]
[[Image:20170209-Plate1-5.png|600px]]
'''B Plate (Samples 6-10)'''


[[Image:20170209-Plate6-10.png|600px]]
[[Image:20170209-Plate6-10.png|600px]]
===Corrections===
'''Plate 2'''
*Switch 9B-T4 and 10A-T4 (D4 and D5)
'''Plate 3'''
*D5: 5A-T3
*D6: 1A-T4
*D7: 1B-T4
*D8: 5B-T3
'''Plate 5'''
*E6: 5A-T4
*E7: 4B-T4
'''Plate 6'''
*J1-Bad
*J8-NTCT
[[Category:ComboLock]] [[Category:20170209]]

Latest revision as of 19:22, 14 February 2017

Beads Binding Test (Started Yesterday)[edit]

Back to Calendar

Protocol-Part 2-qPCR[edit]

  1. qPCR
    1. Make qPCR master mixes according to following recipes
    2. Master Mix Recipes
      Master Mix NoX Forward Primer Amt (10 uM) Reverse Primer Amt (10 uM) uL nfH2O uL 2X Kapa SYBR Fast Total
      73 73 73 1168 1460 2774
      63 63 63 1008 1260 2394
      2.2 2.2 2.2 35.2 44 83.6

      Plate Organization

      Plate Master Mix Quantity Samples Target Primer Pair
      1 73 1-5 Template CLv3-AmpF/CLv3-AmpR
      2 63 6-10 Template CLv3-AmpF/CLv3-AmpR
      3 73 1-5 C1 primer24/primer4RC
      4 63 6-10 C1 primer24/primer4RC
      5 73 1-5 C2 primer6/primer12RC
      6 63 6-10 C2 primer6/primer12RC
    3. Add 38 uL master mix to each well
    4. Add 2 uL sample according to table in following section
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 15 sec
      5. plate read
      6. goto b x40
      7. 72C 2 min
      8. 16C hold

Plate Layouts[edit]

A Plate (Samples 1-5)

File:20170209-Plate1-5.png

B Plate (Samples 6-10)

File:20170209-Plate6-10.png

Corrections[edit]

Plate 2

  • Switch 9B-T4 and 10A-T4 (D4 and D5)

Plate 3

  • D5: 5A-T3
  • D6: 1A-T4
  • D7: 1B-T4
  • D8: 5B-T3

Plate 5

  • E6: 5A-T4
  • E7: 4B-T4

Plate 6

  • J1-Bad
  • J8-NTCT