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=Overload FISH= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] From the results [[Daniel:Notebook/HiResChrPaint/2014-2-26#Sample C (Cot1-HighMW-546 + Cot1-LowMW-488)|last week]], I believe that the probe sets may need an increased signal in order to be visualized. In that spirit, I'm going to amplify more probe and use a lot of it on one experiment. ==qPCR== {| 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="15" | V6S2 (6.1x) | align="center" | 2.44 | align="center" | 305 | align="center" | 2.44 | align="center" | 2.44 | align="center" | 15.25 | align="center" | 282.43 | align="center" | 610 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S3 (6.1x) | align="center" | 2.44 | align="center" | 305 | align="center" | 2.44 | align="center" | 2.44 | align="center" | 15.25 | align="center" | 282.43 | align="center" | 610 |} ===qPCR Results=== [[Image:2014-03-05-V6S2V6S3.png|500px]] ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" valign="bottom" | width="65" height="15" | Sample | width="65" | V6S2A | width="65" | V6S2B | width="65" | V6S3A | width="65" | V6S3B |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | ng/uL DNA | align="center" | 78.9 | align="center" | 77.5 | align="center" | 85.2 | align="center" | 86.2 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 3.2 | align="center" | 3.1 | align="center" | 3.4 | align="center" | 3.4 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | ug total | align="center" | | align="center" | 6.3 | align="center" | | align="center" | 6.9 |} ==Slide Preparation== #Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI #Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI #Allow cells to adhere for 6 hours at 37 C #Rinse slides briefly in 1X PBS #Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS #Rinse briefly in 1X PBS #Incubate for 5 minutes in 1 mL of 2X SSCT #Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide #Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm #Slides are typically best used within 1-2 weeks of creation [[Category:HRCP]] [[Category:20140305]] [[Category:Slide Preparation]] [[Category:qPCR]]
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