Difference between revisions of "Team:UMaryland/protocols"

Revision as of 10:24, 18 September 2015

Miniprep

Materials:

Procedure:

- 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)
- Resuspend pellet in 250 µL Buffer P1
- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear
- Do not allow reaction to proceed for more than 5 mins
- If using Lyse Blue, reagent, solution will turn blue
- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
- Centrifuge for 10 mins at 13000 rpm
- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting
- Centrifuge for 60 secs and discard flow through
- Wash the Q1A prep column with 750 µL Buffer PE
- Centrifuge for 60 secs and discard flow through
- Centrifuge for 60 secs to remove residual wash buffer
- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column
- Let stand for 1 min and centrifuge for 1 min
- Repeat steps 12 and 13

Ligation

Materials:

Procedure:

Transformation

Materials:

Procedure:

@@ Line 210: / Line 210: @@
    <li>- Incubate at 37°</li>
+</ul>
+<br>
+<br>
+</div>
+<div id='contentbox'>
+<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
+<b>3A Assembly</b></a>
+<p style="font-size:24px;text-align:left;text-decoration: underline;">
+Materials:
+<ul class="a">
+  <li>- 5 µL CutSmart</li>
+  <li>- 0.5 µL BSA </li>
+  <li>- 0.5 µL upstream digest (DH5alpha)</li>
+  <li>- 0.5 µL downstream digest </li>
+  <li>- Add DDH2O to 20 µL (DH5alpha)</li>
+</ul>
+<p style="font-size:24px;text-align:left;text-decoration: underline;">
+Procedure:
+<ul class="a">
+  <li>- Combine reagents in a PCR tube</li>
+  <li>- Place in the thermocycler</li>
+  <li>- 37° for 30 minutes</li>
+  <li>- 80° for 20 minutes</li>
 </ul>
 <br>