Daniel:Notebook/ComboLock/2017-2-10: Difference between revisions
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Add 48 uL master mix to each well
Add 2 uL sample according to table in following section
qPCR Cycles
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<li>Add 48 uL of appropriate master mix and 2 uL sample according to plate layout</li> | |||
<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></ol> | |||
'''Plate Organization''' | '''Plate Organization''' |
Revision as of 19:21, 14 February 2017
Beads Binding Test (Started Yesterday)
Protocol-Part 2-qPCR
- qPCR
- Make qPCR master mixes according to following recipes Master Mix Recipes
- Add 48 uL of appropriate master mix and 2 uL sample according to plate layout
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 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 |
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 15 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
Plate Layouts
A Plate (Samples 1-5)
B Plate (Samples 6-10)
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