User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/15

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Plan

 * Miniprep pTXB1His and send for sequencing
 * Start more cultures of new colonies for more minipreps
 * Don't forget to miniprep pTXB1 (unmodified) as well
 * Streak expression strain (ER2566) on LB so competent cells and a glycerol stock can be made

Experiments

 * 1) Glycerol stock
 * 2) * 750μL pTXB1His/DH10B QC colony #1 culture from yesterday + 250 μL 60% glycerol (sterile) &rarr;  tube #79
 * 3) *&rarr; -80°C
 * 4)  Miniprep of pTXB1His
 * 5) follow  Miniprep vacuum protocol for pTXB1His/DH10B QC colony #1 culture from  yesterday
 * 6) Quantify [pTXB1His miniprep #1]
 * 7) 95μL H2O
 * 8) + 5μL miniprepped DNA from step 2
 * 9) * &rarr; measure absorbance at 260 nm and 280 nm
 * 10) DNA sequencing
 * 11) * send pTXB1His #1 for sequencing with M13-Rev and M13-For primers
 * 12) * Also sent Kay Wang's MDHI12VR81M plasmid for sequencing with pKK223-3-For and pBAD-Rev primers
 * 13) * Sequencing is done by | Johns Hopkins Synthesis and Sequencing Facility
 * 14) Miniprep cultures of pTXB1His and pTXB1 in DH10B
 * 15) 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #2 (see  Results below)
 * 16) 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #3 (see  Results below)
 * 17) 10mL LB + 10μL 100 mg/mL Amp + pTXB1/DH10B colony (from  last week)
 * 18) *&rarr; 37°C O/N w/ shaking

Transformations

 * Four plates yesterday:
 * 200μL experimental transformation from yesterday: 1 colony
 * &rarr; pTXB1His/DH10B QC colony #2
 * 200μL control transformation from yesterday: 0
 * 800μL experimental transformation re-plated from 2 days ago: 1 colony
 * &rarr; pTXB1His/DH10B QC colony #3
 * 800μL control transformation re-plated from 2 days ago: 0

Miniprep
$$[pTXB1His\#1]=(0.114AU+0.012AU)\times50\frac{\frac{\mu g}{mL}}{AU}\times\frac{100\mu L}{5\mu L}=102\frac{\mu g}{mL}$$

$$\frac{A_{260}}{A_{280}}=\frac{0.114AU+0.012AU}{0.067AU+0.011AU}=1.82$$

Sequencing

 * Kathryn Muratore 13:00, 23 June 2011 (EDT):

| link to alignment


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