Daniel:Notebook/ComboLock/2017-4-5
Jump to navigation
Jump to search
RCA Test (Started March 24)[edit]
Second Strand Synthesis part II[edit]
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
- TBE Gel-PreBgl
- Mix 96 uL 10X TBE and 24 uL 6X dye
- Aliquot 10 uL on to parafilm per sample
- Add 2 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes File:GelLanes-20170405-RCATest-CPLP-PostqPCR-PreBgl.png
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- TBE Gel-PostBgl
- Mix 120 uL 10X TBE and 40 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
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 |
Results[edit]
- 20170405-qPCR-RCATest-CPLP-ssSynthesis2.png
qPCR curves
- 2017-04-05 -RCATest-CPLP-ssSynthesis2-PreBgl.png
Gel image-pre Bgl digestion
- 2017-04-05 -RCATest-CPLP-ssSynthesis2-PostBgl.png
Gel image-post Bgl digestion