User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/05/26

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
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Today's Benchwork

 * 1)  Miniprep of  O/N culture
 * 2) * Follow quick vacuum protocol, except:
 * 3) ** At step 5, added 750 μL instead of 350 μL Neutralization solution. I "corrected" this by adding an additional 200 μL Cell Lysis solution before step 6, thus increasing the total volume.
 * 4) Quantify [  pCMV-SPORT6+BSA ]
 * 5) 95μL H2O
 * 6) + 5μL miniprepped DNA from step 1
 * 7) * &rarr; measure absorbance at 260 nm and 280 nm
 * 8) PCR of BSA for cloning into pTXBI
 * 9) *diluted pCMV-SPORT6+BSA to 10 μg/mL
 * 10) **5 μL plasmid + 74 μL sterile H2O
 * 11) *followed Basic PCR protocol
 * 12) **template = 10 μg/mL pCMV-SPORT6+BSA
 * 13) **5' primer = 4 μM BSA-NheI-5'
 * 14) **3' primer = 4 μM BSA-SapI-3'
 * 15) **Tanneal = 60°C
 * 16) **textend = 2 min
 * 17) **&rarr; 4°C O/N

Results
$$[CMV-SPORT6+BSA]=(0.130AU+0.028AU)\times50\frac{\frac{\mu g}{mL}}{AU}\times\frac{100\mu L}{5\mu L}=158\frac{\mu g}{mL}$$

$$\frac{A_{260}}{A_{280}}=\frac{0.130AU+0.028AU}{0.055AU+0.029AU}=1.88$$


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