User:Neil J. Parikh/Lab Notebook
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Pipetting & Making Solutions |
Pg. 6 |
<![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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Plate Creation & Cell Culture |
Pg. 7 |
<![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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