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===Quantifying Pre-circularized ppMALAT1=== *Alan did padlock probe capture in vitro [[AlanFung:LabNotes/Capturing/2014-1-23#Probe_calculations_ppMALAT1]] '''Samples''' *5ul of 10ng capture product: stored in 4C *9ul of 1000ng capture product: stored in -20C *Positive control: 20ul of 2uM ppMALAT1 (linear ssDNA) = 40pmole x (92*303.7 + 79) pg/pmole = 1.12ug *Negative control: 25ul of dsDNA PCR product from Noi (~1ug) ====Procedure==== '''Purify circular ssDNA with Zymo Kit''' #Bring each sample to 60ul total with H2O #Use Zymo ssDNA/RNA Clean & Concentrate Kit following default protocol #Elute with 15ul H2O '''Quantify with Qubit ssDNA Assay''' #Buffer mix: 6ul reagent + 1194ul buffer = 1200ul #Add 10ul of each standard control to 190ul buffer mix #Add 1ul of each sample to 199ul buffer mix ====Results==== *10ng capture product: too low to detect **minimum detectable is 50pg/ul = 50ng/ml *1000ng capture product: 15.1ug/ml *PosCtrl: 16.0ug/ml *NegCtrl: too low to detect **Repeated qubit on 2/10/14 with 10ul of sample to find 30ng/ml ====Calculations==== *'''PosCtrl:''' 16 ug/ml = 16 ng/ul **15ul * 16 ng/ul = 240ng **240ng/1.12ug = 21.4% yield *'''1000ng:''' 15.1 ug/ml = 15.1 ng/ul **15ul * 15.1 ng/ul = 226.5ng purified circular ssDNA **Starting amount of ppMALAT1 used in capture reaction: 2ul * 1434 ng/ul = 2.868 ug (from Alan's notes) **Fraction used for purification/quantification: 9/15 * 2.868 ug = 1.7208 ug **226.5ng/1.7208ug = '''13.16% yield''' (includes padlock capture step) ***Assuming circular ssDNA has same yield during purification as linear ssDNA (PosCtrl), then '''61.5% of ppMALAT1 is circularized''' **226.5 ng * (nmole/28019.4ng) = 0.00808 nmole = 8.08 pmole **8.08 pmole/15 ul = 0.539 pmole/ul = 0.539 uM ***If none was used for PCR: 0.539 uM * 15/9 = 0.9uM '''How much pre-circularized ppMALAT1 do we need for positive control?''' *1uM seems pretty reasonable considering that is about what we use for fluorescent-dye probes to detect primary rolonies **Theoretically that mean it's more than the number of primary rolonies and even if we can only fix 1/10th of them that's enough for positive control *Need atleast 150ul so we can do 3 wells with positive control *Will do more reactions tomorrow: [[Matt:LabNotes/2014-2-6]]
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