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=Test PA Gel Polymerization with BS(PEG)9= *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== *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== *BS(PEG)9 only cross-links at pH 7-9 **APS buffers solution to pH 8-9 ***Confirmed with pH test strips ==Gel Mix== *In 1.5ml tube *Add APS and TEMED last *Let sit in 1.5ml tube for 30min *Positive Control {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | 40% AB 199:1 Mix | align="right" | 12.5 |- style="font-size:11pt" valign="bottom" | height="14" | 10% BSA | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 81.5 |- style="font-size:11pt" valign="bottom" | height="14" | 5% TEMED | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | 5% APS | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 100 |} *With BS(PEG)9 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | 40% AB 199:1 Mix | align="right" | 12.5 |- style="font-size:11pt" valign="bottom" | height="14" | 10% BSA | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 79.5 |- style="font-size:11pt" valign="bottom" | height="14" | 5% TEMED | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | 5% APS | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | BS(PEG)9 | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 100 |} ==Result== *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
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