Difference between revisions of "Team:UMaryland/protocols"

Revision as of 14:45, 18 September 2015

Protocols

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:

3A Assembly

Materials:

Procedure:

RE Digest

Materials:

Procedure:

Gibson Assembly

Materials:

Procedure:

PCR

Materials:

Procedure:

- Combine reagents in a PCR tube
- Place tube in thermocycler for:
- Initial Denaturation: 98° C for 30 seconds
- 25-35 cycles: 98°C for 5-10 seconds, 45-72°C for 10-30 seconds and 72°C for 15-30 seconds per kb
- Final Extension: 72°C
- Hold 4-10°C

Gel Extract

Procedure:

- cut out gel portions with scalpel
- weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel
- 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
- After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
- Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)
- The color of solution relates to ph indicator in Q3
- Add 1 gel volume of of isopropyl to the sample and mix. This increases yield of DNA fragments between 500 bp and 4KB
- Place a Q1Aquick spin column in a provided 2ml collection tube
- Apply sample to spin column, centrifuge for 1 min to bind dna
- discard flow through
- wash with .75 mL buffer PE in column, centrifuge 1 minute
- Place column in a clean 1.5 microcentrifuge tube
- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min

Fast Protein Liquid Chromatography

Procedure:

- centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm
- Wash FPLC column with equilibration column (X2) with syringe
- Wash out FLPC system with equilibration buffer
- Wash affinity column with equilibration column with syringe
- Wash affinity column with cobalt acetate with syringe
- Wash off excess cobalt with syringe
- Connect affinity column to FPLC system
- Inject supernatan
- Wash FPLC column with equilibration column (X2) with syringe

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-<b>Giscon Assembly</b></a>
+<b>Gibson Assembly</b></a>
 <p style="font-size:24px;text-align:left;text-decoration: underline;">
 Materials: