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=Padlock0301 Dilution Test (Started [[Daniel:Notebook/ComboLock/2016-10-13|yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="4"> <li>Circularization</li> <ol type="A"> <li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li></ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 32.5 uL exonuclease I (20 units/μL) and 6.5 uL exonuclease III (100 units/μL) in 1:1 ratio</li> <li>Add 2 μL of exonuclease I/III mix to reaction</li> <li>Mix the reaction by swirling pipette around the well 5 times</li> <li>Incubate reaction at 37 ºC for 2 hours</li> <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mix</li> <ol type="a"> <li>334.25 uL nfH2O</li> <li>9.55 uL 100 uM AmpF-CLv2</li> <li>477.5 uL Kapa SYBR Fast MM</li></ol> <li>Add 43 uL master mix to each well</li> <li>Add sample and AmpR indexes according to sample table in the next section</li> <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 176 uL TBE, 44 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===qPCR Sample Table=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="109" height="30" | Sample | width="97" | Template Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="79" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF-CLv2 | width="89" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="77" | Total Volume (uL) |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6A | 10 amol | A1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6B | 10 amol | B1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 6C | 10 amol | C1 | align="center" | 20 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8A | 100 zmol | D1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8B | 100 zmol | E1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 8C | 100 zmol | F1 | align="center" | 21 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9A | 10 zmol | G1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9B | 10 zmol | H1 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 9C | 10 zmol | A12 | align="center" | 22 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10A | 1 zmol | B12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10B | 1 zmol | C12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 10C | 1 zmol | D12 | align="center" | 23 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11A | 100 ymol | E12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11B | 100 ymol | F12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 11C | 100 ymol | G12 | align="center" | 24 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12A | 10 ymol | H12 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12B | 10 ymol | A2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample 12C | 10 ymol | B2 | align="center" | 25 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 17.5 | align="center" | 50 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | NTC | align="center" | 0 | C2 | align="center" | 26 | align="center" | 2 | align="center" | 25 | align="center" | 0.5 | align="center" | 5 | align="center" | 19.5 | align="center" | 50 |} ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161014-qPCR-Padlock0301-Dilutions.png|qPCR Curve File:2016-10-14-Padlock0301-Dilutions.png|Gel Image 1 File:2016-10-14-Padlock0301-Dilutions-Gel2.png|Gel Image 2 </gallery> =BSA Ab-Oligo Linkage= <ol> <li>Buffer Prep</li> <ol type="A"><li>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)</li></ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend antibodies at 1 mg/mL in PBS</li> <li>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</li> <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate</li> <li>Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer</li> <li>Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing</li> <li>Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA</li> <li>Transfer antibodies to new column and spin for 3 min at 1000xg</li></ol> <li>Oligonucleotide Activation</li> <ol type="A"> <li>Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA</li> <li>Add 1.3 uL oligonucleotide to tube (or plate)</li> <li>Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C</li> <li>Add 20 uL PBS with 20 mM EDTA <li>Remove excess DTT using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS</li></ol> </ol> I accidentally eluted the Ab fraction into a wash collector (with wash buffer in it), so I'm going to have to repeat the experiment on [[Daniel:Notebook/ComboLock/2016-10-17|Monday]] =Cell Preparation= Prepping U87-MG cells for an experiment. Since I have a potential cold and I want to nurse it, I'll try and do the actual experiment on [[Daniel:Notebook/ComboLock/2016-10-17|Monday]]. <ol> <li>Fix cells</li> <ol type="A"> <li>Pellet cells at 600g for 3 min</li> <li>Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT</li> <li>Wash cells with wash buffer</li> <li>Pellet cells and permeabilize with 2 mL ice-cold methanol for 10 min on ice; split cells into individual 1.5 mL tubes</li> <li>Store cells in -80C</li> </ol> [[Category:ComboLock]] [[Category:20161013]] [[Category:20161014]]
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