Matt:LabNotes/2016-6-28
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Test PA Gel Polymerization with BS(PEG)9[edit]
- Make sure adding BS(PEG)9 doesn't inhibit polymerization
- Check pH of gel to make sure BS(PEG)9 will work
- Not including Acrydite/Amine linker because it is limited in quantity
- One 100nmole order can be used 3-4 times (~$25 per use, ~$100 total)
Calculate molar concentrations in final mix[edit]
- 5% Acrylamide = 700mM
- 5g/100mL
- 71.08 g/mol
- 0.1% APS = 4.4mM
- 100mg/100ml
- 228.18 g/mol
- 0.1% TEMED = 6.67mM
- 2ul/2ml
- 116.24 g/mol
- 775 kg/m^3 = 775 g/L
- Pick concentration for BS(PEG)9:
- BS(PEG)9 = 5mM
- 708.71 g/mol
- After adding 465ul DMSO to stock bottle: 100mg/565ul = 177 g/L = 250mM
- Dilute 50X in final mix just like APS and TEMED: 5mM
- Pick concentration for Acrydite/Amine Oligo Linker
- Ideally ~1mM in final mix but due to only having 61.1 nmol in tube have to use less
- Resuspend 61.1 nmol in 123.2 ul = 500uM
- Dilute 2X in final mix so final concentration: 250uM
pH of gel mix[edit]
- BS(PEG)9 only cross-links at pH 7-9
- APS buffers solution to pH 8-9
- Confirmed with pH test strips
- APS buffers solution to pH 8-9
Gel Mix[edit]
- In 1.5ml tube
- Add APS and TEMED last
- Let sit in 1.5ml tube for 30min
- Positive Control
Component | Volume |
40% AB 199:1 Mix | 12.5 |
10% BSA | 2 |
H2O | 81.5 |
5% TEMED | 2 |
5% APS | 2 |
Total | 100 |
- With BS(PEG)9
Component | Volume |
40% AB 199:1 Mix | 12.5 |
10% BSA | 2 |
H2O | 79.5 |
5% TEMED | 2 |
5% APS | 2 |
BS(PEG)9 | 2 |
Total | 100 |
Result[edit]
- Positive control did not polymerize as much as usual/expected but there was a solid piece of gel in middle of tube
- With BS(PEG)9 did not polymerize as well as positive control but did show signs of polymerization (more viscous and sticky)
- pH strip indicated pH ~8
- Forgot to include picture