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=Probe Prep Test 2= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Purpose: After extensive troubleshooting, I think I am once again ready to try creating the probes, starting from qPCR and hopefully going all the way to staining. My plan is to use only two sets, V4S3 and V6S3 as before, to help me focus better. The key thing that I've learned from the previous tries is that I need more probe. As such, I am modifying the qPCR. First of all I'm going to use, on Dr. Zhang's recommendation, the suggested protocol for qPCR using the KAPA kit. Previously I was using a different protocol that I felt was a bit short. Also, in previous expansion PCRs I have done 8 reactions of 100 uL each for each probe. I'm going to use 12 for each in an effort to increase yields. ==Expansion qPCR== 1. Set up master reactions according to 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" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="65" | 50 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="22" | per rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.1 | align="center" | 46 | align="center" | 100 |- style="font-size:12pt" align="center" | height="22" | V4S3 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 0 | align="center" | 1.5 | align="center" | 610 | align="center" | 1250 |- style="font-size:12pt" align="center" | height="22" | V6S3 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 5 | align="center" | 1.5 | align="center" | 610 | align="center" | 1250 |} 2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions) 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue 6. Incubated overnight ([[Daniel:Notebook/HiResChrPaint/2013-5-11|continued 5/11/2013]])
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