IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/22

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July 22, 2010

Today we ran 2 digestions: one ligation for the decoder and another that is a part of coliroid. The digestions were:

Digestions
1 2 3 4 5
1.0 uL diluted K091101 0.56 uL E0430 1.11 uL CAP plasmid 0.44 uL R0082 0.54 uL B0034
41.5 uL dH2O 41.94 uL dH2O 41.39 uL dH2O 42.06 uL dH2O 41.96 uL dH2O
5 uL NEBuffer 2 5 uL NEBuffer 2 5 uL NEBuffer 2 5 uL NEBuffer 2 5 uL NEBuffer 2
0.5 uL BSA 0.5 uL BSA 0.5 uL BSA 0.5 uL BSA 0.5 uL BSA
1.0 uL EcoRI-HF 1.0 uL XbaI 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF 1.0 uL XbaI
1.0 uL SpeI 1.0 uL PstI 1.0 uL PstI 1.0 uL SpeI 1.0 uL PstI


The digestions were then incubated at 37 C for 15 minutes, and transfered to 80 C for 20 minutes after that. After the incubations I began ligations of the digestions.

Ligations
1 2
11 uL dH2O 11 uL dH2O
2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer
1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase
2.0 uL Digest 1 2.0 uL Digest 4
2.0 uL Digest 2 2.0 uL Digest 5
2.0 uL Digest 3 2.0 uL Digest 3

The ligations were mixed together by flicking the tubes they were in.

The 2 ligations were incubated in room temperature (25 C) for 10 minutes and then incubated in a 80 C bath for 20 minutes.

Transformation: I did the transformations by electroporation of NEB E.coli cells that were competent. I used 50 uL of NEB cells and 3 uL of ligation mixture. I did two different methods: in one method I mixed the ligation solution in the electro-cuvette, and the other I mixed them in a PCR tube then put them into the electro-cuvette. After electroporation, I added 1 mL LB broth (SOC Outgrowth Medium) and incubated them in the 37 C room for 1 hour. Then I took 200 uL of the broth and plated onto CAP plates. So overall I made 4 plates: 2 with ligation A, and 2 with ligation B. I put the electro-cuvettes in the 4 C room.



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