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=C Probe Hybridization test (VIM, Started [[Daniel:Notebook/ComboLock/2016-5-23|5/23/2016]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] From [[Daniel:Notebook/ComboLock/2016-5-24|yesterday]]'s results, I'm going to try the qPCR again. I saved the samples in the 4C, so I just went straight to the qPCR. Hopefully this time I will get more conclusive evidence one way or the other. ==Protocol== <ol type="A"> <li>Set up reaction according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FCD5B4;font-size:12pt" align="center" | width="175" height="45" | Sample | width="65" | Lane | width="65" | Cell Sample | width="65" | Cells | width="88" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM Primer2 | width="65" | 100 uM Primer4-RC | width="65" | 100 uM Primer6 | width="81" | 100 uM Primer12-RC | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (C1) | align="center" valign="bottom" | A1 | align="center" valign="bottom" | S1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 2 (C2) | align="center" valign="bottom" | A2 | align="center" valign="bottom" | S2 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (C1+C2) | align="center" valign="bottom" | A3 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 (No Probe) | align="center" valign="bottom" | A4 | align="center" valign="bottom" | S4 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | No Template | align="center" valign="bottom" | A5 | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Mismatch Primers | align="center" valign="bottom" | A6 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" | height="15" valign="bottom" | No Primers | align="center" valign="bottom" | A7 | align="center" valign="bottom" | S3 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Primers + C Probes | align="center" valign="bottom" | A8 | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 |} <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 x20</li> <li>72C 2 min</li> <li>16C hold</li> </ol> </ol> ===Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:13pt;font-weight:bold" valign="bottom" | width="65" height="17" | Row | width="180" | Sample | width="65" | Cq |- style="font-size:13pt" | height="17" valign="bottom" | A01 | valign="bottom" | Sample 1 (C1) | align="center" align="center" valign="bottom" | 15.16 |- style="font-size:13pt" | height="17" valign="bottom" | B01 | valign="bottom" | Sample 2 (C2) | align="center" align="center" valign="bottom" | 13.55 |- style="font-size:13pt" | height="17" valign="bottom" | C01 | valign="bottom" | Sample 3 (C1+C2) | align="center" align="center" valign="bottom" | 13.45 |- style="font-size:13pt" | height="17" valign="bottom" | D01 | valign="bottom" | Sample 4 (No Probe) | align="center" valign="bottom" | N/A |- style="font-size:13pt" | height="17" valign="bottom" | E01 | valign="bottom" | No Template | align="center" valign="bottom" | N/A |- style="font-size:13pt" | height="17" valign="bottom" | F01 | valign="bottom" | Mismatch Primers | align="center" valign="bottom" | N/A |- style="font-size:13pt" | height="17" valign="bottom" | G01 | valign="bottom" | No Primers | align="center" valign="bottom" | N/A |- style="font-size:13pt" | height="17" valign="bottom" | H01 | valign="bottom" | Primers + C Probes | align="center" valign="bottom" | N/A |} [[Image:CProbeTest_Results2_20160525_VIM.png|500px]] ==Discussion== Once again all the negative controls failed and so did the positive control. However, this time all 3 samples amplified correctly. I'm inclined therefore to call this a successful run, which means I should try the other samples. =Prep for VIM Antibody Test= [[Daniel:Notebook/ComboLock/2016-5-26|Tomorrow]] I plan on beginning the anitbody prep for testing VIM. Today there is some prep work to be done. <ol type="A"> <li>Prep 100 mL of sodium phosphate buffer</li> <ol type="a"> <li>Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O</li> <li>Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O</li> <li>Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)</li> <li>Directions from [http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8303.full?text_only=true Cold Spring Harbor] </ol> <li>Sulfo-SMCC</li> <ol type="a"> <li>Dissolve 50 mg sulfo-SMCC in 990 uL nf H20 and 110 uL 1M phosphate buffer (Conc: 100 mM SulfoSMCC)</li> <li>Aliquot in 100 uL increments</li> <li>Store at -20C with dessicant</li> </ol>
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