Daniel:Notebook/ComboLock/2017-3-17: Difference between revisions
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qPCR
TBE Gel
>Djacobse |
>Djacobse |
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{| 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="200" height=" | | width="200" height="28" | Reagent | ||
| width="85" | Single Rxn | | width="85" | Single Rxn | ||
| width=" | | width="150" | MM1 (23.2X) | ||
| width=" | | width="150" | MM1-2 (20.2X) | ||
| width="150" | MM+ (C1, 4.2X) | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Primer Pair (fwd/rev) | | height="15" valign="bottom" | Primer Pair (fwd/rev) | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | AmpF/AmpR-Ind21 | ||
| align="center" valign="bottom" | AmpF/AmpR-Ind21 | |||
| align="center" valign="bottom" | primer24/primer4* | | align="center" valign="bottom" | primer24/primer4* | ||
Line 25: | Line 27: | ||
| height="15" valign="bottom" | Forward Primer (10 uM) | | height="15" valign="bottom" | Forward Primer (10 uM) | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 23.2 | ||
| align="center" align="center" valign="bottom" | 20.2 | |||
| align="center" align="center" valign="bottom" | 4.2 | | align="center" align="center" valign="bottom" | 4.2 | ||
Line 31: | Line 34: | ||
| height="15" valign="bottom" | Reverse Primer (10 uM) | | height="15" valign="bottom" | Reverse Primer (10 uM) | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 23.2 | ||
| align="center" align="center" valign="bottom" | 20.2 | |||
| align="center" align="center" valign="bottom" | 4.2 | | align="center" align="center" valign="bottom" | 4.2 | ||
Line 37: | Line 41: | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | 21 | | align="center" align="center" valign="bottom" | 21 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 487.2 | ||
| align="center" align="center" valign="bottom" | 424.2 | |||
| align="center" align="center" valign="bottom" | 88.2 | | align="center" align="center" valign="bottom" | 88.2 | ||
Line 43: | Line 48: | ||
| height="15" valign="bottom" | 2X Kapa SYBR Fast | | height="15" valign="bottom" | 2X Kapa SYBR Fast | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 580 | ||
| align="center" align="center" valign="bottom" | 505 | |||
| align="center" align="center" valign="bottom" | 105 | | align="center" align="center" valign="bottom" | 105 | ||
Line 49: | Line 55: | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 48 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 1113.6 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 969.6 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 201.6 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 201.6 | ||
Revision as of 16:01, 17 March 2017
Circularization Comparison (T4 Poly/Ligase; Started Yesterday)
Protocol-Part 6
- Make qPCR master mixes according to following recipe
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170317-T4Circ-PostRCA.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
Reagent | Single Rxn | MM1 (23.2X) | MM1-2 (20.2X) | MM+ (C1, 4.2X) |
Primer Pair (fwd/rev) | NA | AmpF/AmpR-Ind21 | AmpF/AmpR-Ind21 | primer24/primer4* |
Forward Primer (10 uM) | 1 | 23.2 | 20.2 | 4.2 |
Reverse Primer (10 uM) | 1 | 23.2 | 20.2 | 4.2 |
nfH2O | 21 | 487.2 | 424.2 | 88.2 |
2X Kapa SYBR Fast | 25 | 580 | 505 | 105 |
Total | 48 | 1113.6 | 969.6 | 201.6 |
- Mix 200 uL TBE and 50 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc