User:Neil J. Parikh/Lab Notebook: Difference between revisions

Revision as of 15:09, 6 February 2008

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 <p class=MsoTitle style='margin-right:26.1pt'>Pipetting &amp; Making
 Solutions</p>
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 <p class=ExperimentNumber>Pg. 6</p>
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<![if !supportLists]>              I.        <![endif]>Pipetting<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Learned to use pipettes properly<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Make sure to wipe down pipettes between solutions (with Ethanol)<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Change tips every time you go between solutions<o:p></o:p>

<![if !supportLists]>            II.

<![endif]>Solutions<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Task: Make 0.3M KCl<o:p></o:p>

<![if !supportLists]>                                                  i.    <![endif]>Salt (KCl): CAS 7447-40-7<o:p></o:p>

<![if !supportLists]>                                                ii.    <![endif]>MW: 74.56g/mol<o:p></o:p>

<![if !supportLists]>                                               iii.    <![endif]>2.2368 g / 100mL<o:p></o:p>

<o:p> </o:p>

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 <p class=MsoTitle style='margin-right:26.1pt'>Plate Creation &amp; Cell
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<![if !supportLists]>              I.        <![endif]>Steps for creating a plate:<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Dissolve 25g LB in 800mL of H2O<o:p></o:p>

Bring the final volume to 1L in a 2L flask<o:p></o:p>

Add 15g Bactoagar, cover with foil, autoclave for 20-30 minutes<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Bring back to lab, cool to 50°C while stirring<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Add100mg AMP & let swirl until dissolved<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Pour it into the plates (about 2-3mm deep)<o:p></o:p>

<![if !supportLists]>            II.        <![endif]>Cell Culture:<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Use solution of LB with AMP<o:p></o:p>

Ensure to keep the bags sealed to keep everything sterile <o:p></o:p>

*Note once a bag of plates has been opened, the whole thing must be used<o:p></o:p>

The tubes allow the solution to be aerated<o:p></o:p>

The tubes with bacteria should look cloudy upon visual inspection—can be confirmed with cell density measurement (using the Spec)<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Sterilize the autopipette, set to 1mL (make a 2mL culture, however)<o:p></o:p>

Add 2mL of the LB (for a miniprep); you will have to add 1mL, two times<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Label one as the control and another as your experimental sample<o:p></o:p>

*Label the tubes with your Initials, Sample Name (control or experimental) and the date<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Dip the culture scraper in ethanol and flame lightly (2x) to sterilize<o:p></o:p>

<![if !supportLists]>e.    <![endif]>Let the scraper cool for a few seconds<o:p></o:p>

<![if !supportLists]>f.     <![endif]>Scrape off a tiny bit of bacteria off of the plate and place the scraper into the tube and shake around to ensure the bacteria breaks off into the tube<o:p></o:p>

<![if !supportLists]>g.

<![endif]>Set incubator at 37°C overnight and let shake gently for 12-14hrs<o:p></o:p>

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