Difference between revisions of "Team:UMaryland/protocols"
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| − | <div id=' | + | <div id='cover'> |
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| + | <p style="font-size:64px"><b>Protocols</b> | ||
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| − | < | + | </div> |
| + | </div> | ||
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| − | < | + | <div id='contentbox'> |
| − | + | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;"> | |
| − | < | + | <b>Miniprep</b></a> |
| − | + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | |
| − | + | Materials: | |
| − | </ | + | <ul class="a"> |
| − | <p | + | <li>- 250 µL Buffer P1</li> |
| − | + | <li>- 250 µLBuffer P2</li> | |
| − | + | <li>- 350 µL Buffer N3</li> | |
| − | <ul | + | <li>- 750 µL Buffer PE</li> |
| − | + | <li>- 100 µL DDH2O</li> | |
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</ul> | </ul> | ||
| − | <p | + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> |
| − | + | Procedure: | |
| − | < | + | <ul class="a"> |
| − | + | <li>- Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube) by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)</li> | |
| − | + | <li>- Resuspend pellet in 250 µL Buffer P1</li> | |
| − | + | <li>- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear </li> | |
| − | + | <li>- Do not allow reaction to proceed for more than 5 mins</li> | |
| − | + | <li>- If using Lyse Blue, reagent, solution will turn blue</li> | |
| − | + | <li>- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times | |
| − | + | </li> | |
| − | + | <li>- Centrifuge for 10 mins at 13000 rpm</li> | |
| − | + | <li>- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting</li> | |
| − | + | <li>- Centrifuge for 60 secs and discard flow through</li> | |
| − | + | <li>- Wash the Q1A prep column with 750 µL Buffer PE</li> | |
| − | + | <li>- Centrifuge for 60 secs and discard flow through</li> | |
| − | + | <li>- Centrifuge for 60 secs to remove residual wash buffer</li> | |
| − | + | <li>- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column</li> | |
| − | + | <li>- Let stand for 1 min and centrifuge for 1 min</li> | |
| − | + | <li>- Repeat steps 12 and 13</li> | |
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| − | < | + | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;"> |
| − | + | <b>Ligation</b></a> | |
| − | + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | |
| − | < | + | Materials: |
| − | + | <ul class="a"> | |
| − | + | <li>- 2 µL PSBIA3 digest</li> | |
| − | + | <li>- 2 µL upstream digest (pBAD/ sRNBC)</li> | |
| − | + | <li>- 2 µLdownstream digest (miraculin / const_GFP)</li> | |
| − | + | <li>- 1 µL T4 DNA ligase</li> | |
| − | + | <li>- 2 µL T4 DNA ligase 10x rxn buffer</li> | |
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</ul> | </ul> | ||
| − | <p | + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> |
| − | + | Procedure: | |
| − | + | <ul class="a"> | |
| − | <ul | + | <li>- Combine reagents in a clean PCR tube</li> |
| − | + | <li>- Let stand 10 mins at room temperature/ no heat kill</li> | |
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| − | + | <div id='contentbox'> | |
| + | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;"> | ||
| + | <b>Transformation</b></a> | ||
| + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
| + | Materials: | ||
| + | <ul class="a"> | ||
| + | <li>- 50 µL cells (DH5alpha)</li> | ||
| + | <li>- 2 µL DNA </li> | ||
| + | </ul> | ||
| + | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
| + | Procedure: | ||
| + | <ul class="a"> | ||
| + | <li>- Add DNA to cells</li> | ||
| + | <li>- Incubate on ice for 30 minutes</li> | ||
| + | <li>- Heat shock at 42° fro 30 seconds</li> | ||
| + | <li>- Add 1 mL SOC media to the cells</li> | ||
| + | <li>- Incubate for 60 minutes at 37°</li> | ||
| + | <li>- Plate 200 µL </li> | ||
| + | <li>- Incubate at 37°</li> | ||
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| + | </ul> | ||
| + | <br> | ||
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Revision as of 10:15, 18 September 2015
