PROTOCOLS.html

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<div class="wpmd">
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>PLASMID DNA ISOLATION - BY MINI PREP PROTOCOL&nbsp; </B></font><font color="#969696" face="Calibri" class="ws14"><B> </B></font><font face="Calibri" class="ws14"><B> </B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><B><BR></B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Clean the benches and pipettes.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Centrifuge 1mL of bacterial culture (in log phase of growth) for 7 minutes&nbsp; at&nbsp; 13000rpm in a microcentrifuge.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Pour off the supernatant, leaving abput 50 - 100uL.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Resuspend the bacterial pellet with 300uL of P1 buffer (50mM Tris-HCl, 10uM EDTA and 10ug/mL of RNAse)</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">5.&nbsp;&nbsp;&nbsp; Add 300uL of P2 buffer (0.2 M NaOH, 1% SDS) and mix by inversion. </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">6.&nbsp;&nbsp;&nbsp; Incubate the tubes for 5 minutes at room temperature.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">7.&nbsp;&nbsp;&nbsp; Add 300uL of P3 buffer (3M potassium acetate solution (pH 5.5)), this solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in a insoluble white, rubbery precipitate.&nbsp; </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">8.&nbsp;&nbsp;&nbsp; Mix by inversion.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">9.&nbsp;&nbsp;&nbsp; Place on ice during 5 minutes.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">10.&nbsp;&nbsp;&nbsp; Centrifuge for 10 minutes at 13000rpm.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">11.&nbsp;&nbsp;&nbsp; Carefully, transfer the supernatant to a new microcentrifuge tube and discard the pellet.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">12.&nbsp;&nbsp;&nbsp; Add 600uL of Phenol: chloroform: isoamyl alcohol (24:1:1).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">13.&nbsp;&nbsp;&nbsp; Mix by inversion and centrifuge for 5 minutes at 13000rpm.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">14.&nbsp;&nbsp;&nbsp; Transfer the aqueous phase (?700uL) to new properly labeled microtube.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">15.&nbsp;&nbsp;&nbsp; Add an equal volume of cold isopropanol (?700uL) to the recovered supernatant. Mix by inverting the tube.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">16.&nbsp;&nbsp;&nbsp; Pellet the DNA by centrifugation at 4°C for 15 minutes at 13000rpm.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">17.&nbsp;&nbsp;&nbsp; Pour off isopropanol, being carefully not to loose pellet. Use a pipette tip to remove remaining isopropanol without dislodging the DNA pellet.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">18.&nbsp;&nbsp;&nbsp; Rinse the pellet with 500uL of 70% ethanol. Mix by inversion and centrifuge briefly (5 minutes at 13000rpm).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">19.&nbsp;&nbsp;&nbsp; Remove the residual ethanol and dry the DNA in the DNA SpeedVac.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">20.&nbsp;&nbsp;&nbsp; Resuspend the DNA in 30uL of ultrapure water.</font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>POLYMERASE CHAIN REACTION (PCR) - FOR AMPLIFICATION OF RH1AB GENE COMPLEX</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Clean the bench and the pipettes.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Thawed the PCR mix, nuclease-free water, the primers, and the DNA samples. Place them on ice.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Mix the reagents in each PCR tube, in the following order: (If you planned to make a "Master Mix", calculate the volumes for the amount of samples plus 1 tube). Finally add 2uL of the DNA isolate.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14"><B>Reagent</B></font><font face="Calibri" class="ws14">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </font><font face="Calibri" class="ws14"><B>Volume for 1 tube </B></font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">Nuclease free water&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5.5uL</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">Platinum® Blue PCR Super Mix 2X&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 12.5uL</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">rht 1bF forward primer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.5uL</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">Rht 2bR Reverse primer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2.5uL</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">Premix volume (per tube)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 23uL</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">Amount of DNA sample (per tube)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2uL</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Turn the thermal cycler on and set the Protocol: rh1AB.amp (within iGEM user).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">5.&nbsp;&nbsp;&nbsp; Put the PCR tubes on the thermal cycler. Insert volume data and begin the program.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">6.&nbsp;&nbsp;&nbsp; When the equipment reaches the last temperature setting (at 4°C), take off the tubes and turn off the equipment. </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">7.&nbsp;&nbsp;&nbsp; Use PCR products immediately. Otherwise, keep them at 4°C if they will be used within the first 24 hours or at -20°C to keep for longer periods.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>AGAROSE GEL ELECTROPHORESIS</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Assemble the gel casting tray and comb. The comb should not touch the bottom of the tray.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Add 0.7g of agarose to 70 mL of 1X TAE Buffer. Using a microwave, melt the agarose solution (for 1 minute).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; When the agarose solution has cooled (to about 50°C), add 2uL of ethidium bromide solution and mix it well.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Pour solution directly into the casting tray, ensuring that no bubbles get into the gel. (rinse the flask immediately).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">5.&nbsp;&nbsp;&nbsp; Allow the gel to cool. It will solidify and become slightly opaque within 20 to 30 minutes. Remove the gel casting and set in the correct position.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">6.&nbsp;&nbsp;&nbsp; Submerge the gel by adding approximately 800mL of 1X TAE running buffer to cover the gel by about a half a centimeter.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">7.&nbsp;&nbsp;&nbsp; Carefully remove the comb by lifting it gently at one end, tilting the comb as it comes out. Pulling the comb straight up creates a vacuum in the wells, which tends to lift the whole gel out of the tray. Ensure that the wells are submerged and filled with buffer. </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">8.&nbsp;&nbsp;&nbsp; Prepare the 2uL DNA ladder (marker) for loading using 2uL of loading buffer.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">9.&nbsp;&nbsp;&nbsp; Load a maximum of 10uL of each sample into individual wells with a gel loading tip.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">10.&nbsp;&nbsp;&nbsp; Once all the samples are loaded place the cover on the gel apparatus. Connect the leads so that the red (positive) lead is at the end of the gel&nbsp; to wich DNA will migrate and the&nbsp; black (negative) lead is is at the side of the gel containing the wells. Turn on the power supply and set at 100V. Check the gel after minutes. </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">11.&nbsp;&nbsp;&nbsp; When the blue tracking dye (which runs in these gels along with a DNA fragment of about 200-400bp) has migrated about 50% of the distance to the end of the gel (usually within 60 minutes), turn off the power supply and disconnect the power leads.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">12.&nbsp;&nbsp;&nbsp; Transfer the gel to the transilluminator and visualize DNA with UV light. The DNA fragment band corresponding rhlAB gene complex has 2343bp. </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>TRANSFORMATION</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Thaw the competent cells on ice for 5 minutes (competent cells are extremely susceptible to heat, work with them always on ice.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Meanwhile the cells are thawing out, add 2-4uL of DNA to a 2 mL microtube on ice (use molecular grade H2O for your negative control).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Add 50uL of the thawed out competent cells to 2mL microtube.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Incubate 5 minutes on ice.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">5.&nbsp;&nbsp;&nbsp; Add 200uL of room temperature SOC medium to the microtubes. (It is no longer necessary to keep the cells on ice from this step onwards).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">6.&nbsp;&nbsp;&nbsp; Incubate for 1 - 2 hours at 37°C with vigorous shaking.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">7.&nbsp;&nbsp;&nbsp; Spread 100uL to the transformants onto an agar plate with the appropriate antibiotic.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">8.&nbsp;&nbsp;&nbsp; Incubate at 37°C "overnight" with the top side looking downwards</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>AMPICILLIN STOCK</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Weight the appropriate amount of antibiotic powder for the amount of H20dd to be used. (we use 0.25g in 5mL of H20 to make a 50mg/ml solution).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Absorb your solution with the sterile syringe.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Filter your solution into the sterile 15mL centrifuge tube.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">PREPARING CHEMICALLY COMPETENT CELLS</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Grow an overnight cell culture to the strain you want to turn competent.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Make a 1:100 dilution of the overnight cell culture and grow to a OD600 of 0.3 - 0.5 (we inoculate 1mL of overnight into 100 mL of LB)</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Split the cell culture into two 50 ml centrifuge tubes.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Centrifuge at 4000rpm at 4°C for 10 minutes (Always keep the cells on ince from this point onward, this is very important).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">5.&nbsp;&nbsp;&nbsp; Discard the supernatant.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">6.&nbsp;&nbsp;&nbsp; Gently resuspend the cells using 20mL of cold 0.1M CaCl2 solution for each centrifuge tube.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">7.&nbsp;&nbsp;&nbsp; Incubate on ice for 30 minutes.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">8.&nbsp;&nbsp;&nbsp; Centrifuge at 4000rpm at 4°C for 10 minutes</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">9.&nbsp;&nbsp;&nbsp; Discard supernatant.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">10.&nbsp;&nbsp;&nbsp; Resuspend each tube using 3mL of cold CaCl2 0.1M 15% glycerol solution.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">11.&nbsp;&nbsp;&nbsp; Aliquot however you seem fit in 2mL microtubes or cryovials.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">12.&nbsp;&nbsp;&nbsp; Store at -80°C as soon as you are done.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>AGAR PLATES</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Mix 20g of agar powder for every 1000mL of LB Broth being used.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Autoclave </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Wait till the temperature has gone down to a point where it won't burn to touch and add the appropriate antibiotic at the desired concentration.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Serve 25ml of agar + LB&nbsp; Solution per petri dish.</font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>GLYCEROL STOCK</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Grow an overnight cell culture of the desired sample.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Add 1ml of overnight culture into a 2mL microtube or cryovial.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Add 1mL of 40% glycerol solution into the same tube.</font></div>
 +
<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Store at -80°C (To reactivate simple inoculate 50uL into fresh LB Broth with the appropriate antibiotic).</font></div>
 +
<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>RESTRICTION DIGEST</B></font></div>
 +
<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Quickly vortex all ingredients (Buffer, BSA, DNA from Miniprep) before beginning </font></div>
 +
<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Add the following in a microcentrifuge tube:</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; 5uL of Buffer (NEBuffer2).</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; 1uL of BSA.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; 0.5 picomoles DNA, normally uses 10uL of miniprep or 5uL of purified PCR product.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Water to make 48uL</font></div>
 +
<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Vortex enzymes and add 1uL of each to the tube.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; If you are digesting purified PCR products, add 1uL of Dpnl to the reaction.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Incubate reaction a 37°C water bath for at least one hour.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; If your digesting a "vector", add 1uL Antarctic Phosphatase and 6uL of Phosphatase buffer after 2 hour of incubation and incubate for another hour.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; See Bio-Brick Assembly Schedule for more details.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; 5 Heat kill the digest for 20 minutes at 80°C. </font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">4.&nbsp;&nbsp;&nbsp; Sore digested DNA in the refrigerator (4°C) for use in the very near future.&nbsp;&nbsp; </font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div>
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<div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>LIGATION</B></font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">1.&nbsp;&nbsp;&nbsp; Mix together the following:</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">a.&nbsp;&nbsp;&nbsp; 11uL of deionized water.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">b.&nbsp;&nbsp;&nbsp; 2uL of Reaction Buffer for T4 Ligase.</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">c.&nbsp;&nbsp;&nbsp; 2uL digested "Vector" (Phosphatased).</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">d.&nbsp;&nbsp;&nbsp; 4uL digested "Insert" (PCR product digested with Dpnl).</font></div>
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<div align=justify style="margin-left:40px;line-height:1.50;"><font face="Calibri" class="ws14">e.&nbsp;&nbsp;&nbsp; 1uL of DNA ligase.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">2.&nbsp;&nbsp;&nbsp; Incubate at room temperature for 30 minutes (or 4°C overnight for blunt ends).</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">3.&nbsp;&nbsp;&nbsp; Kill Reaction for 15 minutes at 65°C.&nbsp; </font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14">Notes: If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.</font></div>
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<div align=justify style="line-height:1.50;"><font face="Calibri" class="ws14"><BR></font></div>
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<div><font color="#99CC00" face="Berlin Sans FB" class="ws16">To see our Notebook </font></div>
 +
<div><font color="#99CC00" face="Berlin Sans FB" class="ws16">Click on the book</font></div>
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<div><font face="Berlin Sans FB" class="ws16"><BR></font></div>
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Revision as of 21:23, 19 September 2012

PLASMID DNA ISOLATION - BY MINI PREP PROTOCOL 

1.    Clean the benches and pipettes.
2.    Centrifuge 1mL of bacterial culture (in log phase of growth) for 7 minutes  at  13000rpm in a microcentrifuge.
3.    Pour off the supernatant, leaving abput 50 - 100uL.
4.    Resuspend the bacterial pellet with 300uL of P1 buffer (50mM Tris-HCl, 10uM EDTA and 10ug/mL of RNAse)
5.    Add 300uL of P2 buffer (0.2 M NaOH, 1% SDS) and mix by inversion.
6.    Incubate the tubes for 5 minutes at room temperature.
7.    Add 300uL of P3 buffer (3M potassium acetate solution (pH 5.5)), this solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in a insoluble white, rubbery precipitate. 
8.    Mix by inversion.
9.    Place on ice during 5 minutes.
10.    Centrifuge for 10 minutes at 13000rpm.
11.    Carefully, transfer the supernatant to a new microcentrifuge tube and discard the pellet.
12.    Add 600uL of Phenol: chloroform: isoamyl alcohol (24:1:1).
13.    Mix by inversion and centrifuge for 5 minutes at 13000rpm.
14.    Transfer the aqueous phase (?700uL) to new properly labeled microtube.
15.    Add an equal volume of cold isopropanol (?700uL) to the recovered supernatant. Mix by inverting the tube.
16.    Pellet the DNA by centrifugation at 4°C for 15 minutes at 13000rpm.
17.    Pour off isopropanol, being carefully not to loose pellet. Use a pipette tip to remove remaining isopropanol without dislodging the DNA pellet.
18.    Rinse the pellet with 500uL of 70% ethanol. Mix by inversion and centrifuge briefly (5 minutes at 13000rpm).
19.    Remove the residual ethanol and dry the DNA in the DNA SpeedVac.
20.    Resuspend the DNA in 30uL of ultrapure water.

POLYMERASE CHAIN REACTION (PCR) - FOR AMPLIFICATION OF RH1AB GENE COMPLEX

1.    Clean the bench and the pipettes.
2.    Thawed the PCR mix, nuclease-free water, the primers, and the DNA samples. Place them on ice.
3.    Mix the reagents in each PCR tube, in the following order: (If you planned to make a "Master Mix", calculate the volumes for the amount of samples plus 1 tube). Finally add 2uL of the DNA isolate.
Reagent                                                                     Volume for 1 tube
Nuclease free water                                               5.5uL
Platinum® Blue PCR Super Mix 2X                     12.5uL
rht 1bF forward primer                                        2.5uL
Rht 2bR Reverse primer                                        2.5uL
Premix volume (per tube)                                    23uL
Amount of DNA sample (per tube)                    2uL

4.    Turn the thermal cycler on and set the Protocol: rh1AB.amp (within iGEM user).
5.    Put the PCR tubes on the thermal cycler. Insert volume data and begin the program.
6.    When the equipment reaches the last temperature setting (at 4°C), take off the tubes and turn off the equipment.
7.    Use PCR products immediately. Otherwise, keep them at 4°C if they will be used within the first 24 hours or at -20°C to keep for longer periods.

AGAROSE GEL ELECTROPHORESIS

1.    Assemble the gel casting tray and comb. The comb should not touch the bottom of the tray.
2.    Add 0.7g of agarose to 70 mL of 1X TAE Buffer. Using a microwave, melt the agarose solution (for 1 minute).
3.    When the agarose solution has cooled (to about 50°C), add 2uL of ethidium bromide solution and mix it well.
4.    Pour solution directly into the casting tray, ensuring that no bubbles get into the gel. (rinse the flask immediately).
5.    Allow the gel to cool. It will solidify and become slightly opaque within 20 to 30 minutes. Remove the gel casting and set in the correct position.
6.    Submerge the gel by adding approximately 800mL of 1X TAE running buffer to cover the gel by about a half a centimeter.
7.    Carefully remove the comb by lifting it gently at one end, tilting the comb as it comes out. Pulling the comb straight up creates a vacuum in the wells, which tends to lift the whole gel out of the tray. Ensure that the wells are submerged and filled with buffer.
8.    Prepare the 2uL DNA ladder (marker) for loading using 2uL of loading buffer.
9.    Load a maximum of 10uL of each sample into individual wells with a gel loading tip.
10.    Once all the samples are loaded place the cover on the gel apparatus. Connect the leads so that the red (positive) lead is at the end of the gel  to wich DNA will migrate and the  black (negative) lead is is at the side of the gel containing the wells. Turn on the power supply and set at 100V. Check the gel after minutes.
11.    When the blue tracking dye (which runs in these gels along with a DNA fragment of about 200-400bp) has migrated about 50% of the distance to the end of the gel (usually within 60 minutes), turn off the power supply and disconnect the power leads.
12.    Transfer the gel to the transilluminator and visualize DNA with UV light. The DNA fragment band corresponding rhlAB gene complex has 2343bp.

TRANSFORMATION

1.    Thaw the competent cells on ice for 5 minutes (competent cells are extremely susceptible to heat, work with them always on ice.
2.    Meanwhile the cells are thawing out, add 2-4uL of DNA to a 2 mL microtube on ice (use molecular grade H2O for your negative control).
3.    Add 50uL of the thawed out competent cells to 2mL microtube.
4.    Incubate 5 minutes on ice.
5.    Add 200uL of room temperature SOC medium to the microtubes. (It is no longer necessary to keep the cells on ice from this step onwards).
6.    Incubate for 1 - 2 hours at 37°C with vigorous shaking.
7.    Spread 100uL to the transformants onto an agar plate with the appropriate antibiotic.
8.    Incubate at 37°C "overnight" with the top side looking downwards

AMPICILLIN STOCK

1.    Weight the appropriate amount of antibiotic powder for the amount of H20dd to be used. (we use 0.25g in 5mL of H20 to make a 50mg/ml solution).
2.    Absorb your solution with the sterile syringe.
3.    Filter your solution into the sterile 15mL centrifuge tube.
PREPARING CHEMICALLY COMPETENT CELLS
1.    Grow an overnight cell culture to the strain you want to turn competent.
2.    Make a 1:100 dilution of the overnight cell culture and grow to a OD600 of 0.3 - 0.5 (we inoculate 1mL of overnight into 100 mL of LB)
3.    Split the cell culture into two 50 ml centrifuge tubes.
4.    Centrifuge at 4000rpm at 4°C for 10 minutes (Always keep the cells on ince from this point onward, this is very important).
5.    Discard the supernatant.
6.    Gently resuspend the cells using 20mL of cold 0.1M CaCl2 solution for each centrifuge tube.
7.    Incubate on ice for 30 minutes.
8.    Centrifuge at 4000rpm at 4°C for 10 minutes
9.    Discard supernatant.
10.    Resuspend each tube using 3mL of cold CaCl2 0.1M 15% glycerol solution.
11.    Aliquot however you seem fit in 2mL microtubes or cryovials.
12.    Store at -80°C as soon as you are done.

AGAR PLATES

1.    Mix 20g of agar powder for every 1000mL of LB Broth being used.
2.    Autoclave
3.    Wait till the temperature has gone down to a point where it won't burn to touch and add the appropriate antibiotic at the desired concentration.
4.    Serve 25ml of agar + LB  Solution per petri dish.

GLYCEROL STOCK

1.    Grow an overnight cell culture of the desired sample.
2.    Add 1ml of overnight culture into a 2mL microtube or cryovial.
3.    Add 1mL of 40% glycerol solution into the same tube.
4.    Store at -80°C (To reactivate simple inoculate 50uL into fresh LB Broth with the appropriate antibiotic).

RESTRICTION DIGEST
1.    Quickly vortex all ingredients (Buffer, BSA, DNA from Miniprep) before beginning
2.    Add the following in a microcentrifuge tube:
1.    5uL of Buffer (NEBuffer2).
2.    1uL of BSA.
3.    0.5 picomoles DNA, normally uses 10uL of miniprep or 5uL of purified PCR product.
4.    Water to make 48uL
3.    Vortex enzymes and add 1uL of each to the tube.
1.    If you are digesting purified PCR products, add 1uL of Dpnl to the reaction.
4.    Incubate reaction a 37°C water bath for at least one hour.
1.    If your digesting a "vector", add 1uL Antarctic Phosphatase and 6uL of Phosphatase buffer after 2 hour of incubation and incubate for another hour.
2.    See Bio-Brick Assembly Schedule for more details.
3.    5 Heat kill the digest for 20 minutes at 80°C.
4.    Sore digested DNA in the refrigerator (4°C) for use in the very near future.  

LIGATION
1.    Mix together the following:
a.    11uL of deionized water.
b.    2uL of Reaction Buffer for T4 Ligase.
c.    2uL digested "Vector" (Phosphatased).
d.    4uL digested "Insert" (PCR product digested with Dpnl).
e.    1uL of DNA ligase.
2.    Incubate at room temperature for 30 minutes (or 4°C overnight for blunt ends).
3.    Kill Reaction for 15 minutes at 65°C. 
Notes: If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

To see our Notebook
Click on the book

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