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=qPCR= [[Daniel Jacobsen|Back to Main]] qPCR is different for every experiment, both in how you make master mixes and cycle number/times/temperatures. However, this page has the basics of qPCR and also template qPCR protocols for my experiments. ==Basic qPCR Protocol== Basic qPCR uses these ingredients: *DNA Template *Primers (forward and reverse) *KAPA SYBRFast Master Mix *H2O to bring to reaction volume #Basic thermocycler protocol: ##Initial Denaturation (95C, >1 min) ##Denaturation (95C, 3-10 sec) ##Annealing (50C-60C), 20-40 sec) ##Extension (~72C, 15-40 sec) ##plate read (reads amt DNA present) ##repeat 2-4, (10-30 times) ##Final extension (~72C, >1 min) ##Cool (kill) reaction, 16C hold ==Assorted qPCR Protocols== ==High Resolution Chromosome Painting== #Reaction table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | |style="font-weight:bold" width="65" | 10 nM OligoPool |style="font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM |style="font-weight:bold" width="65" | 100 uM AP1V61U |style="font-weight:bold" width="65" | 100 uM AP2V6 |style="font-weight:bold" width="65" | 2 mM aa-dUTP |style="font-weight:bold" width="65" | H20 |style="font-weight:bold" width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 0.4 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 2.5 | align="center" | 46.3 | align="center" | 100 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | BSPP 1 (xxNxx.1x) | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |} #Aliquot 100 uL into each of xxNxx qPCR tubes #PCR protocol: ##PCR 3 min 95C ##95C 3 sec ##55C 30 sec ##72C 20 sec ##plate read ##goto ii x17 ##72C 2 min ##16C hold #Pool 3 samples together #Column Purification, elute with 40 uL EB #Measure concentrations in nanodrop ==Presets== <ol> <li>"Standard" protocol (used for most things)</li> <ol type="a"> <li>95C 2 min</li> <li>95C 20 sec</li> <li>58C 30 sec</li> <li>70C 30 sec</li> <li>Goto b X times (Variable, usually about 40)</li> <li>4C forever</li> </ol></ol>
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