DNA Quantification – NcoI Linearized RLP Plasmid DNA from Yesterday
Performed DNA quantification according to Friedman Lab protocol using Pico Green and the Teacan plate reader.
R2 = 0.99989
y = 662.22x + 624.42
Plate results (mean concentration in ng/uL) are here. Standards on the plate used were 0, 1, 5, 10, 20 ng/uL.
Quanting standard curve looks spectacular.
NcoI linearized RLP plasmid = 18.21 ng/uL
Will make a fresh RLP plasmid curve and check with qPCR.
Upon attempting to prepare the dilutions using one of Nate’s dilution calculation sheets, I realized that the dilution sheets that I looked at were inconsistent in regards to the size of the linerarized RLP plasmid! The sheet that Lisa most recently used (2/29/2012) indicates that the RLP plasmid is 5454bp in size! This is definitely incorrect as can be seen by my gel from yesterday (the linearized fragment doesn’t run greater than 5000bp. Additionally, Nate has other calculation sheets that indicate the plasmid size is 4133bp! This seems to jive both with my gel results from yesterday. However, this suggests that the last curve that Lisa made was incorrect because the calculations were based off of an incorrect plasmid size.
This discrepancy could explain the shift in the most recent curves to coming up at later Cqs than what Nate’s old curve had been coming up at, since using the plasmid size of 5454bp would assume you had more copies of plasmid than were actually there. Thus, the calculated copy number would actually be less than expected at each dilution point of the plasmid curve.
Will need to discuss with Lisa to find out exact size of plasmid with RLP insert.
20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.