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=" | | width="85" height="45" | Master Mix NoX | ||
| width=" | | width="105" | Forward Primer Amt (10 uM) | ||
| width="105" | Reverse Primer Amt (10 uM) | |||
| width="85" | uL nfH2O | |||
| width=" | | width="105" | uL 2X Kapa SYBR Fast | ||
| width=" | | width="85" | Total | ||
| width=" | |||
| width="65" | | |- 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" | | | height="15" valign="bottom" | 1 | ||
| 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" valign="bottom" | 1-5 | ||
| | | align="center" valign="bottom" | C1 | ||
| | | align="center" valign="bottom" | primer24/primer4RC | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | 4 | ||
| align="center" | | align="center" align="center" valign="bottom" | 63 | ||
| align="center" valign="bottom" | 6-10 | |||
| | | align="center" valign="bottom" | C1 | ||
| | | align="center" valign="bottom" | primer24/primer4RC | ||
| | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | 5 | ||
| align="center" | | align="center" align="center" valign="bottom" | 73 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | 1-5 | ||
| align="center" align="center" valign="bottom" | | | align="center" valign="bottom" | C2 | ||
| | | align="center" valign="bottom" | primer6/primer12RC | ||
| align="center" align="center" valign="bottom" | | |||
| | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| | | 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 | <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 | <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]
Protocol-Part 2-qPCR[edit]
- qPCR
- Make qPCR master mixes according to following recipes Master Mix Recipes
- Add 38 uL master mix to each well
- Add 2 uL sample according to table in following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 15 sec
- plate read
- goto b x40
- 72C 2 min
- 16C hold
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 |
Plate Layouts[edit]
A Plate (Samples 1-5)
B Plate (Samples 6-10)
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