Daniel:Notebook/ComboLock/2017-4-3
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RCA Test (Started March 24)[edit]
I'll try qPCR on the RCA product again, this time on the NEB 12 hr samples (since I am running low on X samples), but for few cycles and with only the forward primer (which should be RC to the RCA product. This will be fed into Bglii digestion
- qPCR-Second strand synthesis
- Make the following 13.2X master mix
- 13.2 uL AmpF-6.4Sol (10 uM)
- 264 uL Kapa SYBR 2X Master Mix
- 224.4 uL nfH2O
- Aliquot 38 uL master mix into each lane
- Add 2 uL sample according to plate layout File:PlateLayout-20170403-RCATest-CPLP-ssSynthesis.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
Protocol-Post-Bglii samples[edit]
- TBE Gel
- Mix 80 uL TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2.5 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
- Qiaquick Column
- Add 200 uL (5X) PB buffer to 40 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 35 uL nfH2O to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- TBE Gel
- Mix 40 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE ladder
- Add 6 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
Results[edit]
- 20170403-qPCR-RCATest-CPLP-ssSynthesis.png
qPCR curves
- 2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii.png
Pre-purification gel
- 2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii-Purified.png
Post purification gel
Purification really didn't do much but it the gels look good. The product should be around 200bp, btw, because I only used AmpF, which adds on about 20bp
Protocol-Pre-Bglii samples[edit]
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- TBE Gel
- 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
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 8 | 0 |
10X Buffer 3.1 | 2 | 12.4 |
Bglii | 1 | 0 |
nfH2O | 9 | 55.8 |
Total | 20 | 68.2 |
File:2017-04-04 -RCATest-CPLP-ssSynthesis-sssThenBgl.png
It appears that the Bgl digestion was incomplete. That does mean there is a lot more product available than can be cut in an hour with one uL of Bglii, which according to NEB is 10 ug (1 ug/unit/hr)