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   			<p class="text-3 p3">
Preparation of Electrocompetent Cells for E. coli

<p class="p4">•         Inoculate 500 ml of L-broth with a fresh overnight E. coli culture</p> 
<p class="p4">•         Grow cells at 37 degrees C shaking at 300 rpm to OD600 of 0.5-0.7</p> 
<p class="p4">•         Chill cells on ice for ~20 mins. For the rest of the steps keep cells as close to 0 degrees as possible including chilling all containers before adding cells. To harvest transfer cells to cold centrifuge bottle and spin at 4000xg for 15 mins at 4 degrees Celsius</p>  
<p class="p4">•         Carefully pout and discard supernatant. Better to pour off a few cells than leaving supernatant behind.</p> 
<p class="p4">•         Resuspend the pellet in 500 ml of ice cold 10% glycerol. Centrifuge at 4000xg for 15 mins at 4 degrees Celsius, pour off and discard supernatant</p> 
<p class="p4">•         Resuspend in 250 ml of ice cold 10% glycerol. Centrifuge at 4000xg for 15 mins at 4 degrees Celsius. Pour off and discard supernatant.</p>  
<p class="p4">•         Resuspend the pellet in a final volume of 1-2 ml of ice cold glycerol. The cell concentration should be 1-3 x 1010 cells/ml</p> 
<p class="p4">•         May be stored in aliquots on dry ice at -70 degrees Celsius. (Stable for 6 months)</p> 

<p class="p4">LB broth</p> 
<p class="p4">Add the following to 800ml H2O</p>
<p class="p4">10g Bacto-tryptone</p>
<p class="p4">5g yeast extract</p>
<p class="p4">10g NaCl</p>
<p class="p4">Adjust pH to 7.5 with NaOH</p>
<p class="p4">Adjust volume to 1L with dH2O</p>
<p class="p4">Sterilize by autoclaving</p>
<p class="p4">Once cooled, add zeocin 250 ul</p>


<p class="p4">YPD Media</p>
<p class="p4">Dissolve 10g of BactoYeast extract in 500ml water</p>
<p class="p4">Dissolve 20g of BactoPeptone in the above solution</p>
<p class="p4">Dissolve 20g Dextrose in the above solution</p>
<p class="p4">Add 20g agar</p>
<p class="p4">Melt agar into solution in the microwave</p>
<p class="p4">fill to 1000ml with water</p>
<p class="p4">Autoclave</p>

        <p class="p4">Polymerase chain reaction using designed primers to isolate of genes from Pichia pastoris.</p>
<p class="p4">PCR is the relatively inexpensive procedure for the amplification of a single or a few segments of DNA using forward and reverse primers, taq polymerase, and a buffer solution.  Add the master mix to a PCR tube. The primers have been designed to insert appropriate restriction sites that will make it an iGEM compatible biobrick. After mixing the following component, put the tube in the thermocycler and start the following cycle.</p>
 <p class="p4"> Gel electrophoresis to determine if the correct gene was isolated</p>
<p class="p4">0.42 of Agarose in 35 mL of TAE</p>
<p class="p4">Add 3.5uL of biogreen</p>
<p class="p4">Heat in the microwave for 60 seconds and then pour in a tray.</p>
<p class="p4">Run gel at 90V for 45 minutes</p>
<p class="p4">Image gel using PC Image on computer in 481</p>
                 <p class="p4">Quantification of DNA</p>
<p class="p4">Use the nanodrop in 613 Kell Hall to measure the DNA</p>
<p class="p4">Check for 260/280 ratio, should be about 1.8 for DNA</p>
                
 <p class="p4">Restriction disgestion</p>
<p class="p4">Fermentas fast digest enzymes were used for restriction digestion. The reaction volumes vary based upon the concentration of DNA used and whether you are cutting plasmid DNA or PCR product.</p> 

<p class="p4">For PCR</p>
<p class="p4">Add 25uL of water to all the vials, Add 2uL of 10X buffer, Add 1uL of respective PCR product up to ug</p>
<p class="p4">Add 1uL of restriction enzyme, Mix gently and incubate at 37 degrees C for 30 minutes</p>

<p class="p4">For Plasmid</p>
<p class="p4">Add 15uL of water, Add 2uL of 10X fast digest buffer, Add2uL of DNA (up to 1ug), Add 1uL of restriction enzyme, Spin down, Incubate at 37 degrees C for 5 minutes, Inactivate thermally or through precipitation of DNA</p>

 <p class="p4">Used the protocol indicated in QIAGEN manual for Midi and Mini prep (bold indicates measurements for midi prep)</p>
Notes before starting
•          Add RNase A solution to Buffer P1, mix, and store at 2–8°C.
<p class="p4">•         Optional: Add LyseBlue® reagent to Buffer P1 at a ratio of 1:1000.</p>
<p class="p4">•          Prechill Buffer P3 at 4°C. Check Buffer P2 for SDS precipitation.</p>
<p class="p4">•          Isopropanol and 70% ethanol are required.</p>
<p class="p4">•          Symbols:  QIAGEN Plasmid Mini Kit;  QIAGEN Plasmid Midi Kit</p>
<p class="p4">Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min  at 4°C.</p>
<p class="p4">1.     Resuspend the bacterial pellet in  0.3 ml,4 ml</p>
<p class="p4">2.     Add  0.3 ml, 4 ml Buffer(bold)'P2, mix thoroughly by vigorously inverting 4–6 times, and incubate at room temperature (15–25°C) for 5 min.  If using LyseBlue reagent, the solution will turn blue.</p>
<p class="p4">3.      Add 0.3 ml, 0.5 ml(bold) prechilled Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice            5 min,  15 min If using LyseBlue reagent, mix the solution until it is colorless.</p>
<p class="p4">4.     Centrifuge at 14,000–18,000 x g for 10 min at 4°C. Re-centrifuge if the supernatant is not clear.
Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the supernatant at ≥20,000 x g for 15 min at 4°C.</p>
      <p class="p4">5.     Equilibrate a QIAGEN-tip  20, 100 by applying 1 ml,
4 ml Buffer(bold) QBT, and allow column to empty by gravity flow.</p>
      <p class="p4">6.     Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter
              the resin by gravity flow.</p> 
      <p class="p4">7.    Wash the QIAGEN-tip with  2 x 2 ml, 2 x 10 ml(bold) Buffer QC. Allow Buffer QC to move through the  
             QIAGEN- tip by gravity flow.</p>
      <p class="p4">8.    Elute DNA with  0.8 ml, 5 ml(bold) Buffer QF into a clean
              2 ml,  15 ml, vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to    increase the yield.</p>
      <p class="p4">9.    Precipitate DNA by adding  0.56 ml, 3.5 ml(bold) room-temperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.</p>
    <p class="p4">10.   Wash the DNA pellet with 1 ml, 2 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10   min. Carefully decant supernatant.</p>
    <p class="p4">11. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris•Cl, pH 8.5).</p>

           
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