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==Testing== Doing some QC control on the antibody experiment to figure out what went wrong. The first thing to test is the BSA Ab-oligo complexes. <ol> <li>qPCR</li> <ol type="A"> <li>Make master mixes according to following recipes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FABF8F;font-size:12pt;font-weight:bold" align="center" | width="105" height="30" | Master Mix | width="65" | No. Samples | width="100" | Primers | width="120" | uL Reverse Primer (10 uM) | width="120" | uL Forward Primer (10 uM) | width="85" | uL Kapa SYBR Fast | width="85" | uL nfH2O |- style="font-size:12pt" | height="15" valign="bottom" | PA | align="center" align="center" valign="bottom" | 10 |style="font-weight:bold" align="center" | p24/p4RC | align="center" align="center" valign="bottom" | 10.1 | align="center" align="center" valign="bottom" | 10.1 | align="center" align="center" valign="bottom" | 252.5 | align="center" align="center" valign="bottom" | 212.1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PB | align="center" align="center" valign="bottom" | 2 |style="font-weight:bold" align="center" | p6/p12RC | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 52.5 | align="center" align="center" valign="bottom" | 44.1 |- style="font-size:12pt" | height="15" valign="bottom" | Nonsense A | align="center" align="center" valign="bottom" | 2 |style="font-weight:bold" align="center" | p24/p12rc | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 52.5 | align="center" align="center" valign="bottom" | 44.1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Nonsense B | align="center" align="center" valign="bottom" | 2 |style="font-weight:bold" align="center" valign="bottom" | p6/p4RC | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 2.1 | align="center" align="center" valign="bottom" | 52.5 | align="center" align="center" valign="bottom" | 44.1 |} <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample according to table in following 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 160 uL TBE, 40 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> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161104-qPCR-QC-AntibodyTest-PosNeg.png|qPCR curve comparing positive controls (BSA) vs. negative controls (NTC) File:20161104-qPCR-QC-AntibodyTest-TimeCourse.png|Time course comparison (negative controls included) File:2016-11-04-AntibodyTest-QC-Gel1.png|Gel Image 1 File:2016-11-04-AntibodyTest-QC-Gel2.png|Gel Image 2 </gallery> ===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="105" height="45" | Sample | width="105" | Primers | width="85" | Primer Target | width="65" | Time Point | width="65" | Lane | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="85" | 10 uM Forward Primer | width="85" | 10 uM Reverse Primer | width="65" | H2O | width="105" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | BSA-Ab |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | NA | align="center" | A1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | BSA-Ab |style="font-weight:bold" align="center" | p6/p12RC |style="font-weight:bold" align="center" | PB |style="font-weight:bold" align="center" | NA | align="center" | A2 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | BSA-Ab |style="font-weight:bold" align="center" | p24/p12rc |style="font-weight:bold" align="center" | Nonsense |style="font-weight:bold" align="center" | NA | align="center" | A3 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | BSA-Ab |style="font-weight:bold" align="center" valign="bottom" | p6/p4RC |style="font-weight:bold" align="center" valign="bottom" | Nonsense |style="font-weight:bold" align="center" valign="bottom" | NA | align="center" | A4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample1A |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T1 | align="center" | A5 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample1B |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T1 | align="center" | A6 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample2A |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T2 | align="center" | A7 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample2B |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" valign="bottom" | PA |style="font-weight:bold" align="center" valign="bottom" | T2 | align="center" | A8 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample3A |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T3 | align="center" | H1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample3B |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" valign="bottom" | PA |style="font-weight:bold" align="center" valign="bottom" | T3 | align="center" | H2 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample4A |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T4 | align="center" | H3 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample4B |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | T4 | align="center" | H4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | NTC |style="font-weight:bold" align="center" | p24/p4RC |style="font-weight:bold" align="center" | PA |style="font-weight:bold" align="center" | NA | align="center" | H5 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | NTC |style="font-weight:bold" align="center" | p6/p12RC |style="font-weight:bold" align="center" | PB |style="font-weight:bold" align="center" | NA | align="center" | H6 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | NTC |style="font-weight:bold" align="center" | p24/p12rc |style="font-weight:bold" align="center" | Nonsense |style="font-weight:bold" align="center" | NA | align="center" | H7 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | NTC |style="font-weight:bold" align="center" valign="bottom" | p6/p4RC |style="font-weight:bold" align="center" valign="bottom" | Nonsense |style="font-weight:bold" align="center" | NA | align="center" | H8 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |} [[Category:ComboLock]] [[Category:20161103]]
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