Daniel:Notebook/ComboLock/2016-11-16
Jump to navigation
Jump to search
BSA Beads Test (Started Yesterday)[edit]
Protocol[edit]
- Rolling Circle Amplification
- Prepare 10.2X Master mix according to table below
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make master mix according to following recipe
- 422.1 uL nfH2O
- 502.5 uL SYBR Fast
- 20.1 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 200 uL TBE, 50 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Reagent | uL Added | Master Mix (10.2X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 25.5 |
dNTP (1 mM) | 0.8 | 8.16 |
10X Buffer | 2 | 20.4 |
Phi29 | 1 | 10.2 |
nfH2O | 8.7 | 88.74 |
Total | 20 | 153 |
Results[edit]
- 20161116-qPCR-BSABeads-ChrisPrimer.png
qPCR curve; Chris' RCA primer
- 20161116-qPCR-BSABeads-DanPrimer.png
qPCR curve; Dan's RCA primer
- 2016-11-16-RCA-BSA-ChrisPrimer.png
Gel image 1; Chris' RCA Primer
- 2016-11-16-RCA-BSA-DanPrimer.png
Gel image 2; Dan's RCA Primer
qPCR Sample Table[edit]
Sample | RCA-Primer | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1AC | Chris | 2 ug BSA-biotin | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BC | Chris | 2 ug BSA-biotin | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AC | Chris | No BSA-biotin | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BC | Chris | No BSA-biotin | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AC | Chris | No Antibody | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BC | Chris | No Antibody | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AC | Chris | 20 ng BSA-biotin | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BC | Chris | 20 ng BSA-biotin | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5AC | Chris | 200 pg BSA-biotin | D1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5BC | Chris | 200 pg BSA-biotin | D2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AD | Dan | 2 ug BSA-biotin | D3 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BD | Dan | 2 ug BSA-biotin | D4 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AD | Dan | No BSA-biotin | D5 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BD | Dan | No BSA-biotin | D6 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AD | Dan | No Antibody | D7 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BD | Dan | No Antibody | D8 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AD | Dan | 20 ng BSA-biotin | H1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BD | Dan | 20 ng BSA-biotin | H2 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5AD | Dan | 200 pg BSA-biotin | H3 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | H4 | 25 | 0 | 25 | 1 | 1 | 23 | 50 |
Library Prep (Started Yesterday; Exp. Start 11/7/16)[edit]
- Ethanol Precipitation
- Centrifuge for 25 minutes at 12000 rpm at 4C
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge for 12 minutes at 12000 rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A and B by reusing the water
Sample | ng/uL | ug in 30 uL |
Cells RCA Product (2AB,2BB) | 47.8 | 1.4 |
DEJ-CL-CellC01-20161116-Ind25