BioAnalyzer:
←Older revision
Revision as of 20:39, 6 May 2014
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*Remember to deduct the total from the signup sheet on the computer room door.
*Remember to deduct the total from the signup sheet on the computer room door.
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==Sequencing at the Core==
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1. Get EXO, SAP, and Dilution Buffer tubes from “EXO SAP” box in mini freezer. Keep EXO and SAP on ice to prevent enzymes from denaturing. <br>
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2. Flick or pipette mix EXO and SAP – do not vortex. Make up master mix: <br>
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<u>EXOSAP Master Mix (1 reaction in 1 direction)</u>
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<table border="1">
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<tr>
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<td><b>Reagents</b></td>
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<td><b>1x</b></td>
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</tr>
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<tr>
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<td>dH<sub>2</sub>O</td>
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<td>1μL</td>
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</tr>
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<tr>
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<td>Dilution Buffer</td>
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<td>0.25μL</td>
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</tr>
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<tr>
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<td>EXO</td>
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<td>0.25μL</td>
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</tr>
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<tr>
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<td>SAP</td>
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<td>0.5μL</td>
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</tr>
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<tr>
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<td>PCR Product</td>
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<td>3.0μL</td>
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</tr>
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<tr>
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<td>TOTAL</td>
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<td>5.0μL</td>
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</tr>
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</table>
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3. Aliquot 2uL master mix into PCR tubes or 96-well PCR plate.<br>
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4. Combine with 3uL PCR product per tube/well. Pipette mix.<br>
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5. Spin down tubes/plate and run EXO SAP program on PCR machine (~1hr):<br>
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* 37°C for 30 min (to perform digestion)<br>
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* 80°C for 10 min (inactivate the enzymes)<br>
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* 4°C forever<br>
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6. Store cleaned PCR product in freezer (-20) until ready to set up BIG DYE reaction. <br>
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<br>
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BIG DYE:<br>
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1. Get Big Dye from mini freezer. Keep on ice and cover while thawing (light sensitive).<br>
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2. Get Seq Saver from refrigerator.<br>
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3. Make up master mix:<br>
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<u>BIG DYE Master Mix (1 reaction in 1 direction)</u>
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<table border="1">
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<tr>
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<td><b>Reagents</b></td>
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<td><b>1x</b></td>
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</tr>
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<tr>
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<td>dH<sub>2</sub>O</td>
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<td>3μL</td>
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</tr>
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<tr>
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<td>Seq Saver</td>
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<td>0.5μL</td>
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</tr>
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<tr>
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<td>Big Dye (Light Sensitive!)</td>
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<td>0.5μL</td>
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</tr>
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<tr>
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<td>Primer (3μM)</td>
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<td>1.0μL</td>
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</tr>
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<tr>
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<td>Clean PCR Product</td>
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<td>5.0μL</td>
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</tr>
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<tr>
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<td>TOTAL</td>
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<td>10.0μL</td>
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</tr>
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</table>
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* The volume of PCR product can vary from 1.5-5ul depending on how concentrated your PCR product is. Remember to adjust the volume of water if you adjust the volume of PCR product. <br>
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4. Aliquot 5uL master mix into PCR tubes or 96-well PCR plate.<br>
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5. Combine with 5uL PCR product per tube/well. Pipette mix contents.<br>
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6. Spin down tubes/plate and run BIGDYE05 program on PCR machine:<br>
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* 96 degrees for 10 sec <br>
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* 96 degrees for 10 sec <br>
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* 50 degrees for 20 sec <br>
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* 60 degrees for 4 min <br>
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** 35x of b, c, d
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* 4 degrees forever<br>
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7. Store cycle sequencing product in freezer (-20) until ready to send to Core. <br>
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<br>
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<u>Sequencing sign up</u><br>
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1. Sign up to sequence at [http://www.genoseq.ucla.edu/webseq Genoseq CORE].<br>
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2. Click on R2R Signup (list) (found on the left hand side) and fill out required information. <br>
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3. Answer YES for request cleaning service. <br>
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4. Bring the given reference number with you, check in your samples when you drop them off, and bring back sample racks. Alternatively, you can bring your samples over to the Core and sign up there. <br>
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*Carry samples on ice to the Core.<br>
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==BioAnalyzer==
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# Quantify samples (use Nanodrop, Qubit, or PicoGreen) and record concentration.
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# Using dH2O, dilute 1ul of sample to ~ 2.5-3 ng/uL. If using Nanodrop numbers, use 5-10 ng/uL to account for error. The total final volume must be at least 3ul.
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# Take samples on ice to Core (CHS 36-125). Sign up on the sheet and place samples on a rack. Label the rack and store in freezer.
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# To check for results, log into [https://www.genoseq.ucla.edu/webseq/ WebSeq] and click "Genotyping" on the left side menu.
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Notes:
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*Maximum number of samples for DNA High Sensitivity and RNA Pico chips is 11. Other chips are 12 samples.
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*Try to fill up chip as much as possible by coordinating with other labs.
==Printing field labels==
==Printing field labels==