Difference between revisions of "Team:UMaryland/protocols"

Latest revision as of 02:32, 19 September 2015

Miniprep

Materials:

- 250 µL Buffer P1
- 250 µLBuffer P2
- 350 µL Buffer N3
- 750 µL Buffer PE
- 100 µL DDH2O

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:

- 2 µL PSBIA3 digest
- 2 µL upstream digest (pBAD/ sRNBC)
- 2 µLdownstream digest (miraculin / const_GFP)
- 1 µL T4 DNA ligase
- 2 µL T4 DNA ligase 10x rxn buffer

Procedure:

- Combine reagents in a clean PCR tube
- Let stand 10 mins at room temperature/ no heat kill

Transformation

Materials:

- 50 µL cells (DH5alpha)
- 2 µL DNA

Procedure:

- Add DNA to cells
- Incubate on ice for 30 minutes
- Heat shock at 42°C fro 30 seconds
- Add 1 mL SOC media to the cells
- Incubate for 60 minutes at 37°C
- Plate 200 µL
- Incubate at 37°C

@@ Line 329: / Line 329: @@
    <li>- Heat shock at 42°C fro 30 seconds</li>
    <li>- Add 1 mL SOC media to the cells</li>
-   <li>- Incubate for 60 minutes at 37°</li>
+   <li>- Incubate for 60 minutes at 37°C</li>
    <li>- Plate 200 µL </li>
    <li>- Incubate at 37°C</li>
@@ Line 441: / Line 441: @@
 <ul class="a">
    <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>- 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
   </li>
@@ Line 451: / Line 451: @@
    <li>- Place a Q1Aquick spin column in a provided 2ml collection tube</li>
    <li>- Apply sample to spin column, centrifuge for 1 min to bind dna</li>
-   <li>- discard flow through</li>
+   <li>- Discard flow through</li>
-   <li>- wash with .75 mL buffer PE in column, centrifuge 1 minute</li>
+   <li>- Wash with .75 mL buffer PE in column, centrifuge 1 minute</li>
    <li>- Place column in a clean 1.5 microcentrifuge tube</li>
    <li>- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min</li>
@@ Line 466: / Line 466: @@
 Procedure:
 <ul class="a">
-   <li>- centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li>
+   <li>- Centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li>
    <li>- Wash FPLC column with equilibration column (X2) with  syringe</li>
    <li>- Wash out FLPC system with equilibration buffer</li>