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.

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