Difference between revisions of "Team:UMaryland/protocols"

Revision as of 16:46, 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 306: / Line 306: @@
 Procedure:
 <ul class="a">
-   <li>- cut out gel portions with scalpel</li>
+   <li>- Cut out gel portions with scalpel</li>
    <li>- weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel</li>
    <li>- Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
@@ Line 338: / Line 338: @@
    <li>- Wash off excess cobalt with syringe</li>
    <li>- Connect affinity column to FPLC system</li>
-   <li>- Inject supernatan</li>
+   <li>- Inject supernatant</li>
    <li>- Wash FPLC column with equilibration column (X2) with syringe</li>
 </ul>