Daniel:Notebook/ComboLock/2017-4-5: Difference between revisions
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TBE Gel
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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="font-size:12pt;font-weight:bold" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
|style="background-color:#8DB4E2" width=" | |style="background-color:#8DB4E2" width="180" height="30" | Reagent | ||
|style="background-color:#8DB4E2" width=" | |style="background-color:#8DB4E2" width="85" | Single Rxn Vol | ||
|style="background-color:#95B3D7" width=" | |style="background-color:#95B3D7" width="85" | Master Mix (12.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" |
Revision as of 21:58, 5 April 2017
RCA Test (Started March 24)
Second Strand Synthesis part II
Since the second strand synthesis from Monday worked well, I'm going to repeat the experiment but using the NEB 3 hr and 6 hr samples (X and P, respectively). I won't bother with the Bgl digested ones as I'll just digest after.
- qPCR-Second strand synthesis
- Make the following 14.2X master mix
- 14.2 uL AmpF-6.4Sol (10 uM)
- 284 uL Kapa SYBR 2X Master Mix
- 241.4 uL nfH2O
- Aliquot 38 uL master mix into each lane
- Add 2 uL sample according to plate layout File:PlateLayout-qPCR-20170405-RCATest-CPLP-ssSynthesis2.png
- Run the following protocol
- 95C for 2 min
- 95C for 5 sec
- 55C for 30 sec
- 72C for 45 sec
- Goto (b) x20
- 72C for 2 min
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 11 uL master mix into each sample
- Add appropriate sample and 1 uL Bglii to each
- Incubate 1 hr 37C
- Heat kill with 65C for 20 min
Reagent | Single Rxn Vol | Master Mix (12.2X) |
RCA Rxn | 8 | 0 |
10X Buffer 3.1 | 2 | 24.4 |
Bglii | 1 | 0 |
nfH2O | 9 | 111.6 |
Total | 20 | 136 |
- Mix 60 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
- Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc