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==Cell Lysis== [[File:IMG_1123.jpg|thumb|300px]] *I would like to do an alkaline cell lysis with NaOH, after doing some research the required amount of OH- has to be at least 20mM NaOH in PBS (pH 11.7) for quick cell lysis * We will test the pH of the NaOH with litmus paper Range 0-14 (Ordered placed on 9/20) *Add 1ul NaOH to 24ul nuclease free water, test pH with litmus paper (Increase amount of NaOH if the pH is not high enough) *Both pH are at close to 14 *Seems like the NaOH from the kit is basic enough to lyse the cells <div style="float: right;"> <h3>NAOH</h3> *Add 23 uL 0.5 mg/mL BSA to cells and *Add 1 uL Imprint Balance solution to each sample *Incubate at 37 C for 10 minutes *Cells are now lysed</div> <h3>NP40 w Protease</h3> *Add 3 uL Protease to 97 uL H2O for a 3:100 dilution *Add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; * incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes *Cells are now lysed *Add 21 uL 0.5 mg/mL BSA and *Add 1 uL Imprint Balance solution to sample *incubate at 37 C for 10 minutes <div style="float: middle;"> <h3>Bisulfite Conversion - Imprint Kit</h3> * Prepare DNA Modification Solution (CT Conversion Reagent): * Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes * Perform Column Purification of each sample: * Elute into 11uL Elution Buffer </div> <h2>qPCR w bisulfite identification primers</h2> <div style="float: right;"> <h3>Layout</h3> {| {{table}} | NP40-8 |- | NP40-21 |- | NP40-22 |- | NaOH-8 |- | NaOH-21 |- | NaOH-22 |} </div> *Prepare Master Mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''MM''' | align="center" style="background:#f0f0f0;"|'''CHR8''' | align="center" style="background:#f0f0f0;"|'''CHR21''' | align="center" style="background:#f0f0f0;"|'''CHR22''' |- | 2X iQ Super Mix||44||44||44 |- | Primer F (3.3uM)||13.2||13.2||13.2 |- | Primer R (3.3uM)||13.2||13.2||13.2 |- | gDNA||Do not add to MM||Do not add to MM||Do not add to MM |- | H2O||13.2||13.2||13.2 |} * Add 2ul of the modified DNA to a qPCR striptube, add 39ul MM to each tube Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever
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