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

Revision as of 16:51, 6 February 2008

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</head>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:5.4pt;border-collapse:collapse;border:none;mso-border-bottom-alt:
solid windowtext .5pt;mso-yfti-tbllook:191;mso-padding-alt:0in 5.4pt 0in 5.4pt'>
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;mso-yfti-lastrow:yes'>
 <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style1 style='margin-right:26.1pt'>Pipetting &amp; Making
 Solutions</p>  </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber style1>Pg. 6</p>  </td>
</tr>

</table>

<![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>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:5.4pt;border-collapse:collapse;border:none;mso-border-bottom-alt:
solid windowtext .5pt;mso-yfti-tbllook:191;mso-padding-alt:0in 5.4pt 0in 5.4pt'>
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;mso-yfti-lastrow:yes'>
 <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style1 style='margin-right:26.1pt'>Plate Creation &amp; Cell
 Culture</p>  </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber style1>Pg. 7</p>  </td>
</tr>

</table>

<![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>

<o:p> </o:p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:5.4pt;border-collapse:collapse;border:none;mso-border-bottom-alt:
solid windowtext .5pt;mso-yfti-tbllook:191;mso-padding-alt:0in 5.4pt 0in 5.4pt'>
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;mso-yfti-lastrow:yes'>
 <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style1 style='margin-right:26.1pt'>Transformation of E.coli with
 Recombinant DNA<o:p></o:p></p>  </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 background:#333333;mso-shading:windowtext;mso-pattern:gray-80 auto;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber style1>Pg. 9<o:p></o:p></p>  </td>
</tr>

</table>

*note, the heat shock to the tubes is very important for transcription to occur<o:p></o:p>

+ Controls:<o:p></o:p>

BBa_I13522

pTetGFP<o:p></o:p>

BBa_J45220<o:p></o:p>

Group 1:<o:p></o:p>

BBa_R0011<o:p></o:p>

BBa_J45219<o:p></o:p>

Group 2:<o:p></o:p>

BBa_I14032 / BBa_R0051 (same)<o:p></o:p>

BBa_J06702<o:p></o:p>

Group 3:<o:p></o:p>

BBa_J04500<o:p></o:p>

BBa_J04630 / BBa_I13401<o:p></o:p>

<o:p> </o:p>

Rima & I did the transformation for Group 2:<o:p></o:p>

<![if !supportLists]>                              I.        <![endif]>BBa_I14032<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Name: promoter P(Lac) IQ<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Sequence: promoter (-35, -10 bases before coding)<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Location: 2007, Plate 2, Well 17K<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Plasmid: pSB2K3<o:p></o:p>

<![if !supportLists]>                            II.        <![endif]>BBa_R0051<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Name: promoter (lamda cl regulated)<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Sequence: R0051 promoter<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Location: 2007, Plate 1, Well 9C<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Plasmid: pSB1A2<o:p></o:p>

<![if !supportLists]>                           III.        <![endif]>BBa_J06702<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Name: mCherry, bacterial with RBS & forward terminator<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Sequence: <o:p></o:p>

<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0

style='margin-left:159.5pt;border-collapse:collapse;border:none;mso-border-alt:
solid windowtext .5pt;mso-yfti-tbllook:1184;mso-padding-alt:0in 5.4pt 0in 5.4pt'>
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 <td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt;
 mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;mso-bidi-font-style:normal'>RBS<o:p></o:p></span></span></p>
 </td>
 <td width=85 valign=top style='width:64.05pt;border:solid windowtext 1.0pt;
 border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;mso-bidi-font-style:normal'>mCherry<o:p></o:p></span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt;
 border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;mso-bidi-font-style:normal'>Terminator<o:p></o:p></span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt;
 border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;mso-bidi-font-style:normal'>Terminator<o:p></o:p></span></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:1;mso-yfti-lastrow:yes'>
 <td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt;
 border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0034<o:p></o:p></span></span></p>
 </td>
 <td width=85 valign=top style='width:64.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>J06504<o:p></o:p></span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0010<o:p></o:p></span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
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 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0012<o:p></o:p></span></span></p>
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<![if !supportLists]>c.    <![endif]>Location: 2007, Plate 2, Well 11D<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Plasmid: pSB1A2<o:p></o:p>

<![if !supportLists]>                          IV.        <![endif]>Cells Used: E.coli: JM109 (Promega); Catalog #: L2001<o:p></o:p>

<o:p> </o:p>

<![if !supportLists]>                           V.        <![endif]>3 Plates Made:<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Control: 100μL LB / Cell<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Test Plate 1: 100μL LB/ Cell<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Test Plate 2: 1μL cell : 99μ H2O<o:p></o:p>

<o:p> </o:p>

Actual Transformation Procedure:<o:p></o:p>

<![if !supportLists]>                              I.        <![endif]>If using DNA from the registry, dilute with 9μL of H2O. If using our own DNA, use 200ng. <o:p></o:p>

<![if !supportLists]>a.                <![endif]>inject the water directly into the well, titerate, and then pipette it out<o:p></o:p>

<![if !supportLists]>                            II.        <![endif]>Thaw 100μL competent cells on ice. (50 μL with our own/ 100 μ if commercial cells used)<o:p></o:p>

<![if !supportLists]>                           III.        <![endif]>Mix competent cells with 50 μ CaCl2 (only if using our own cells)<o:p></o:p>

<![if !supportLists]>                          IV.        <![endif]>Add 4 μL of registry DNA, or 200ng of our own DNA to the cells. <o:p></o:p>

<![if !supportLists]>                           V.        <![endif]>Put in the ice bath for about 20 minutes. <o:p></o:p>

<![if !supportLists]>                          VI.        <![endif]>Remove and immediately put in the hot water bath (42°C) to heat shock for 45-60 sec.<o:p></o:p>

<![if !supportLists]>                         VII.        <![endif]>Place back in the ice bath for 2 minutes. <o:p></o:p>

<![if !supportLists]>                       VIII.        <![endif]>Add 900 μL ice cold, antibiotic free LB to the cells<o:p></o:p>

<![if !supportLists]>                          IX.        <![endif]>Incubate at 37°C for 90 minutes in the shaking incubator.<o:p></o:p>

<![if !supportLists]>                           X.        <![endif]>Dilute as desired, make the necessary controls, and plate cells using 100 μL / plate. (200 if not commercial)<o:p></o:p>

<![if !supportLists]>                          XI.        <![endif]>Incubate upside down (so that the label you wrote on it is face up) at 37°C for 18-24 hours, then refrigerate. <o:p></o:p>

<![if !supportLists]>a.                <![endif]>The controls should not have bacteria growing on them!<o:p></o:p>

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

Revision as of 16:51, 6 February 2008

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