Daniel:Notebook/ComboLock/2017-3-29
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RCA Test (Started March 24)[edit]
Bglii Digestion[edit]
Testing bglii digestion of RCA product. No qPCR to start with (RCA should be enough). For recall, here is the sample matrix again and the RCA conditions used.
Sample | Enzyme | Incubation Time (hrs) |
X (A-F) | NEB | 3 |
Y (A-F) | Thermo | 3 |
Z (A-F) | Epi (1:10) | 3 |
P (A-F) | NEB | 6 |
Q (A-F) | NEB | 12 |
- Bglii 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
- 2 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- TBE gel
- Mix 200 uL TBE and 50 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 1.5 uL reaction or ladder to appropriate aliquot File:20170329-GelLanes-NEB.png File:20170329-GelLanes-TF.png
- 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 (30.2X) |
RCA Rxn | 2 | 0 |
Bglii Cutter Primer + (100 uM) | 2 | 60.4 |
10X Buffer 3.1 | 2 | 60.4 |
Bglii | 1 | 0 |
nfH2O | 13 | 392.6 |
Total | 20 | 513.4 |
Results[edit]
- 2017-03-29-RCATest-CPLP-Bglii-NEB-3hr.png
Gel image-3 hr NEB
- 2017-03-29-RCATest-CPLP-Bglii-TF-3hr.png
Gel image-3 hr Thermo Fisher
Gel amounts might not be right. Too much ladder or too little sample
dsDNA Qubit[edit]
Gonna try a double stranded DNA measurement in Qubit to see if there is any signal.
- Annealing
- Mix two uL reaction, 2 uL 100 uM Bglii + primer, and 6 uL nfH2O for samples XA-XF (NEB 3hr); include one XA with 2 uL 100 uM Bglii - primer
- Incubate at 95C for 5 min
- Incubate at 50C for 10 minutes
- Qubit
- Mix 1990 uL Qubit dsDNA buffer and 10 uL Qubit dsDNA dye
- Add 190 uL mix and 10 uL sample for S1 and S2 standards
- Add 199 uL mix and 1 uL sample for samples; include XA without bglii primer (8 samples total)
- Incubate in dark 5 minutes
- Measure in Qubit; use new calibration
Sample Name | Condition | ug/mL | ng/mL | bp | nM |
XA | NEB RCA 3 hrs CP (LLRC) | 45.5 | 188 | 733.4 | |
XB | NEB RCA 3 hrs CP (LLRC*) | 64 | 188 | 1031.59 | |
XC | NEB RCA 3 hrs CP (LLRC***) | 74.7 | 188 | 1204.06 | |
XD | NEB RCA 3 hrs LP (LLRC) | 65.1 | 188 | 1049.32 | |
XE | NEB RCA 3 hrs LP (LLRC*) | 47.7 | 188 | 768.86 | |
XF | NEB RCA 3 hrs LP (LLRC***) | 54.3 | 188 | 875.24 | |
XA (-) | NEB RCA 3 hrs CP (LLRC) - Primer for antisense (-) strand | 49.2 | 188 | 793.04 | |
XA-No primer | NEB RCA 3 hrs CP (LLRC) No added primer | 2.73 | 188 | 44 |