Difference between revisions of "Team:Carnegie Mellon/Protocols"

1. Centrifuge 1.5 ml of culture for 1min full speed at 15000 rpm.
2. Remove supernatant.
3. Add 250 µl of extraction buffer (1% octyl-beta-thioglucoside in 10mM Tris-Cl, pH7.5).
4. Incubate at room temperature for 10 min.

1. Remove 50 µl of Ni-NTA in 10 mM Trischloride bead solution and place in new tube for each purification.
2. Add 1 mL buffer (see table).
3. Centrifuge at 800 rpm for 30 seconds.
4. Remove supernatant.
5. Repeat 3 times and resuspend in 1 mL of Bead Wash Solution.


Seen below is the protocol we standardized and distributed to labs like the Citizen Science Center in an effort to gain more uniformity between data observed in different labs around the world. The back of the card has details on how to elute the protein off on the washed Ni-NTa beads.

FRONT

BACK

PROTEIN PURIFICATION CARD JPEG

Procedure:

1. Set up overnight cultures for miniprep.
2. Make the following reaction recipe:
   5 mL LB
   5 µL Chlorophenical
   1 µL overnight colony
3. Incubate in 37°C for 16-18 hours.
4. Follow the Life Technologies Miniprep Kit Protocol.

1. Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.

Reagent	Amount (µL)
10X Fast Digest Buffer	2
Plasmid DNA	12
Restriction Enzyme XbaI	1
Restriction Enzyme SpeI	1
Water (to bring up to volume)	2
Total Volume	18

2. Digest at 37 °C for 1 hour.
3. Follow the Life Technologies Restriction Enzyme Digest Protocol.

1. 1% aragose gel made of:
   50 mL 0.5X TAE
   0.5g agarose
   2.5 µL ethidium bromide
2. Run at 114V.

1. Make 250-750 µl binding buffer (depending on mass of gel cut-out).
2. Incubated at 42℃ until gel is melted.
3. Follow Thermo Scientific's GeneJET Gel Extracton Protocol

1. Make the following ligation reaction.

Reagent	Amount (µL)
Promoter DNA	1
Insert DNA	7
Ligation Buffer	1
Ligation Enzyme	1
Total Volume	10

2. Ligate for 10 minutes at room temperature.
3. Put on ice until ready for transformation.

1. Follow iGEM's Transformation Protocol