Daniel:Notebook/ComboLock/2016-10-17: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 313: | Line 313: | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|qPCR image | File:20161017-qPCR-Padlock0301-Dilutions2.png|qPCR image | ||
File:|Gel image 1 | File:|Gel image 1 | ||
File:|Gel image 2 | File:|Gel image 2 |
Revision as of 19:46, 17 October 2016
BSA Ab-Oligo Linkage (From Friday)
Friday I accidentally let the post-sulfo SMCC reaction elute into the wash container, which probably messed things up. So I'm repeating that part of the experiment and will mix the new Ab fraction with the oligo fraction prepared on Friday.
Protocol
- Buffer Prep
- Mix 1.4 mL of monobasic and 3.6 mL of dibasic and 45 mL ddH2O to make 50 mL of 100 mM (buffer pH 7.2)
- Antibody Activation
- Resuspend antibodies at 1 mg/mL in PBS
- Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min
- Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate
- Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer
- Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing
- Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
- Transfer antibodies to new column and spin for 3 min at 1000xg
- Oligonucleotide Activation
- Ab-Oligo Mixing
- Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
- Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
- Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
- Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl
- Store at 4C
Padlock0301 Dilutions Test (Started Thursday 10-13)
I'm going to repeat the PCR test, just in case.
- qPCR
- Prepare the following master mix
- 334.25 uL nfH2O
- 9.55 uL 100 uM AmpF-CLv2
- 477.5 uL Kapa SYBR Fast MM
- Add 43 uL master mix to each well
- Add sample and AmpR indexes according to sample table in the next 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 176 uL TBE, 44 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
qPCR Sample Table
Sample | Template Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CLv2 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 6A | 10 amol | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6B | 10 amol | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6C | 10 amol | C1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8A | 100 zmol | D1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8B | 100 zmol | E1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8C | 100 zmol | F1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9A | 10 zmol | G1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9B | 10 zmol | H1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9C | 10 zmol | A12 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10A | 1 zmol | B12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10B | 1 zmol | C12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10C | 1 zmol | D12 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11A | 100 ymol | E12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11B | 100 ymol | F12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 11C | 100 ymol | G12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12A | 10 ymol | H12 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12B | 10 ymol | A2 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 12C | 10 ymol | B2 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | C2 | 26 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
Results
- 20161017-qPCR-Padlock0301-Dilutions2.png
qPCR image