User:Neil J. Parikh/Lab Notebook

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 I.          Pipetting

a. Learned to use pipettes properly

b. Make sure to wipe down pipettes between solutions (with Ethanol)

<span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c. <span style='color:windowtext; font-weight:normal;font-style:normal'>Change tips every time you go between solutions

<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Solutions

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a. <span style='color:windowtext; font-weight:normal;font-style:normal'>Task: Make 0.3M KCl

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> i.     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Salt (KCl): CAS 7447-40-7

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> ii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>MW: 74.56g/mol

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> iii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>2.2368 g / 100mL

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.          <span style='color:windowtext; font-weight:normal;font-style:normal'>Steps for creating a plate  <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>:

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a. <span style='color:windowtext; font-weight:normal;font-style:normal'>Dissolve 25g LB in 800mL of H2O

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Bring the final volume to 1L in a 2L flask

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Add 15g Bactoagar, cover with foil, autoclave for 20-30 minutes

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>b. <span style='color:windowtext; font-weight:normal;font-style:normal'>Bring back to lab, cool to 50 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>° <span style='color:windowtext;font-weight:normal;font-style:normal'>C <span class=MsoIntenseEmphasis> while stirring

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add100mg AMP &amp; let swirl until dissolved

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>d. <span style='color:windowtext; font-weight:normal;font-style:normal'>Pour it into the plates (about 2-3mm deep)

<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Cell Culture  <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>:

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a. <span style='color:windowtext; font-weight:normal;font-style:normal'>Use solution of LB with AMP

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Ensure to keep the bags sealed to keep everything sterile

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>*Note once a bag of plates has been opened, the whole thing must be used

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>The tubes allow the solution to be aerated

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal'>The tubes with bacteria should look cloudy upon visual inspection—can be confirmed with cell density measurement (using the Spec)

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>b. <span style='color:windowtext; font-weight:normal;font-style:normal'>Sterilize the autopipette, set to 1mL (make a 2mL culture, however)

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Add 2mL of the LB (for a miniprep); you will have to add 1mL, two times

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c. <span style='color:windowtext; font-weight:normal;font-style:normal'>Label one as the control and another as your experimental sample

<p class=MsoNormal style='margin-left:1.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>*Label the tubes with your Initials, Sample Name (control or experimental) and the date

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>d. <span style='color:windowtext; font-weight:normal;font-style:normal'>Dip the culture scraper in ethanol and flame lightly (2x) to sterilize

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>e. <span style='color:windowtext; font-weight:normal;font-style:normal'>Let the scraper cool for a few seconds

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>f. <span style='color:windowtext; font-weight:normal;font-style:normal'>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

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>g. <span style='color:windowtext; font-weight:normal;font-style:normal'>Set incubator at 37 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>° <span style='color:windowtext;font-weight:normal;font-style:normal'>C overnight and let shake gently for 12-14hrs

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.75in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>*note, <span class=MsoIntenseEmphasis> the heat shock <span style='color:windowtext; font-weight:normal;font-style:normal'> to the tubes is very important for transcription to occur

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           + Controls :

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_I13522 : pTetGFP

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_J45220

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           Group 1:

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_R0011

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_J45219

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           Group 2:

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_I14032 / BBa_R0051 (same)

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_J06702

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           Group 3:

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_J04500

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>                       BBa_J04630 / BBa_I13401

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='text-indent:.5in'> <span style='color:windowtext;font-weight:normal'>Rima &amp; I did the transformation for Group 2:

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.         <span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_I14032

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> a.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Name: <span style='color:windowtext; font-weight:normal;font-style:normal'>promoter P(Lac) IQ

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> b.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Sequence: <span style='color:windowtext; font-weight:normal;font-style:normal'>promoter <span class=MsoIntenseEmphasis> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>(-35, -10 bases before coding)

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> c.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Location <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>: 2007, Plate 2, Well 17K

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> d.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Plasmid: <span style='color:windowtext; font-weight:normal;font-style:normal'>pSB2K3

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_R0051

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> a.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Name: <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> promoter (lamda cl regulated)

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> b.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Sequence: <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> R0051 promoter

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> c.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Location: <span style='color:windowtext; font-weight:normal;font-style:normal'>2007, Plate 1, Well 9C

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><i>d.<span style='font:7.0pt "Times New Roman"'>   </i><span class=MsoIntenseEmphasis> Plasmid: <i> </i>pSB1A2

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> III. <span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_J06702

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> a.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Name: <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> mCherry, bacterial with RBS &amp; forward terminator

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> b.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Sequence <span style='color:windowtext;font-weight:normal'>:

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> c.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Location: <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> 2007, Plate 2, Well 11D

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> d.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis> Plasmid: <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> pSB1A2

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> IV. <span style='color:windowtext; font-weight:normal;font-style:normal'>Cells Used: E.coli: JM109 (Promega); Catalog #: L2001

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal'>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> V.         <span style='color:windowtext; font-weight:normal;font-style:normal'>3 Plates Made:

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> a.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Control: 100&#956;L LB / Cell

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> b.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Test Plate 1: 100&#956;L LB/ Cell

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis> c.<span style='font:7.0pt "Times New Roman"'>     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Test Plate 2: 1&#956;L cell : 99&#956; H2O

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Actual Transformation Procedure:

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.         <span style='color:windowtext; font-weight:normal;font-style:normal'>If using DNA from the registry, dilute with 9&#956;L of H2O. If using our own DNA, use 200ng.

<p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a. <span style='color:windowtext; font-weight:normal;font-style:normal'>inject the water directly into the well, titerate, and then pipette it out

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Thaw 100&#956;L competent cells on ice. (50 <span style='color:windowtext; font-weight:normal;font-style:normal'>&#956;L with our own/ 100  <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956; if commercial cells used)

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> III. <span style='color:windowtext; font-weight:normal;font-style:normal'>Mix competent cells with 50 <span style='color:windowtext;font-weight:normal; font-style:normal'>&#956; CaCl2 (only if using our own cells)

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> IV. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 4 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L of registry DNA, or 200ng of our own DNA to the cells.

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> V.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Put in the ice bath for about 20 minutes.

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VI. <span style='color:windowtext; font-weight:normal;font-style:normal'>Remove and immediately put in the hot water bath (42 <span style='color:windowtext;font-weight:normal;font-style:normal'>°  <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>C) to heat shock for 45-60 sec.

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VII. <span style='color:windowtext; font-weight:normal;font-style:normal'>Place back in the ice bath for 2 minutes.

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VIII. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 900 &#956;L ice cold, antibiotic free LB to the cells

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> IX. <span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate at 37 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>° <span style='color:windowtext;font-weight:normal;font-style:normal'>C for 90 minutes in the shaking incubator.

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> X.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Dilute as desired, make the necessary controls, and plate cells using 100 &#956;L / plate. (200 if not commercial)

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> XI. <span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate upside down (so that the label you wrote on it is face up) at 37 <span style='color:windowtext;font-weight:normal;font-style:normal'>° <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>C for 18-24 hours, then refrigerate.

<p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a. <span style='color:windowtext; font-weight:normal;font-style:normal'>The controls should not have bacteria growing on them!

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>*Start with Parts: E0241 (x3), R0010, R0040, C0040 from the registry*

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>E0241: PoPs to GFP Converter

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§ <span style='color:windowtext; font-weight:normal;font-style:normal'>RBS: B0032

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§ <span style='color:windowtext; font-weight:normal;font-style:normal'>GFP: E0040

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§ <span style='color:windowtext; font-weight:normal;font-style:normal'>Terminator: B0016

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>R0010: Promoter Lac I (Regulator)

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>R0040: Promoter (tetR, negative)

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>C0040: Tetracycline repressor from transposon Tn10 (+LVA) [note that +LVA is a degradation tag]

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Look out for:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>            <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Control should not be cloudy at all, culture should have gas bubbles, should be cloudy, especially at the bottom

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           *If you start seeing sediment at the bottom, the cells <span class=MsoIntenseEmphasis> may <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> have lysed and let proteins into the tube*

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='text-indent:.5in'> <span style='color:windowtext;font-weight:normal'>Procedure (General):

<p class=MsoNormal style='margin-left:1.25in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Use the QIAGEN Kit to separate the plasmid DNA (so it should not have much chromosomal DNA)

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Grow cells for 12 to 14 hours

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Suspend cells in

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> i.     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 1 (with glucose &amp; EDTA)

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> ii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 2 (SDS-detergent, NaOH) [SDS gets rid of the membrane and NaOH denatures the plasmid and DNA]

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> iii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 3 (K Acetate / Acetic Acid)

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-things become an insoluble precipitate

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-renature the DNA/plasmid

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-only the plasmid DNA will renature properly

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-the plasmid DNA remains soluble &amp; the chromosomal DNA will become the precipitate

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> III. <span style='color:windowtext; font-weight:normal;font-style:normal'>Columns: use EtOH to extract DNA

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Procedure (Specific):

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           *make sure to check your buffers for salt precipitation—this may indicate a change in concentration*

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Get solution and put 1.5mL into an Eppendorf tube to centrifuge

<p class=MsoNormal style='margin-left:1.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>-after centrifuging for 30 seconds @ 13k RPM, remove the supernatant

<p class=MsoNormal style='margin-left:1.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>-add more solution, and centrifuge again using same procedure

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 250 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L PI to tube after the supernatant is <span class=MsoIntenseEmphasis> fully removed

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> III. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 250 &#956;L P2, and invert 4 to 6 times

<p class=MsoNormal style='margin-left:1.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>-immediately add 350 <span style='color:windowtext; font-weight:normal;font-style:normal'>&#956;L N3, and invert again 4 to 6 times

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> IV. <span style='color:windowtext; font-weight:normal;font-style:normal'>Dispose of the flow through, add 0.5mL of buffer PB (that is <span style='color:windowtext;font-weight:normal'>milliliter not microliter!)  <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> then centrifuge for 30-60 seconds

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> V.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 750 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L PE, then centrifuge again, remove the waste, centrifuge again

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VI. <span style='color:windowtext; font-weight:normal;font-style:normal'>Add 50 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L BE buffer

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VII. <span style='color:windowtext; font-weight:normal;font-style:normal'>Measure the DNA concentration using Nanodrop

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VIII. <span style='color:windowtext; font-weight:normal;font-style:normal'>Ethanol Precipitation <span class=MsoIntenseEmphasis> (see separate procedure)

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal'>

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           *used to purify the DNA (really small fragments) or to increase the concentration of it

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> I.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Add:

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> i.     <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>3 volumes of COLD 100% EtOH

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> ii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1/10 volume of 3M sodium acetate

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> iii. <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1 &#956;L GlycoBlue

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> II. <span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate for 1 hr @ 80 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>° <span style='color:windowtext;font-weight:normal;font-style:normal'>C

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> III. <span style='color:windowtext; font-weight:normal;font-style:normal'>Centrifuge for 30min @ 0 <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>° <span style='color:windowtext;font-weight:normal;font-style:normal'>C

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> IV. <span style='color:windowtext; font-weight:normal;font-style:normal'>Remove supernatant

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> V.         <span style='color:windowtext; font-weight:normal;font-style:normal'>Air Dry

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> VI. <span style='color:windowtext; font-weight:normal;font-style:normal'>Resuspend in H2O

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Specific Procedure Example:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           For 50 &#956;L:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>                       Add 150 &#956;L EtOH

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>                                   20 &#956;L NaAc.

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           For 100 &#956;L:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>                       Add     300 &#956;L EtOH

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>                                   40 &#956;L NaAc.

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           *applying an electric field over the gel will separate fragments by size

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>           *compare the movement of the DNA to the ladder

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>            <span class=MsoIntenseEmphasis> *ensure that the well is on the negative side!*

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>DNA has a (-) charge because of the phosphate groups

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>moves toward the anode (+) and away from the cathode (-)

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>DNA is separated by size, distance ~ 1 / molecular weight

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§ <span style='color:windowtext; font-weight:normal;font-style:normal'>100bp-50kbp

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>Gel acts as a sieve: 0.3% large fragments to 2% small fragments

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>® <span style='color:windowtext; font-weight:normal;font-style:normal'>after the gel is made, put it in the microwave for 1minute, then <span style='color:windowtext;font-weight:normal'>cool the bottle in H­2O, and take the lid off

<p class=MsoNormal> <span style='color:windowtext; font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Casting Gel:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           1% Agarose, 0.5g Agarose

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           50mL TBE + SYBR Safe

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Dilute the 5xTBE to 0.5x

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>Add 2 &#956;L of the loading dye!

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>Ladder Formula:

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal'>            <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1 &#956;L 1kbase ladder

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           2 &#956;L BlueJuice Dye

<p class=MsoNormal style='margin-left:.5in'> <span style='color:windowtext;font-weight:normal;font-style:normal'>           7 &#956;L H2O