IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/29

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July 29, 2010

Another digestion/ligation filled day at the IGB for the RNA Decoder! Here is what went down:

Digestions
1 2 3 4 5 6 7 8 9
1.0 uL diluted J23100 0.48 uL E1010 1.55 uL B0015 1.0 uL diluted E0020 1.0 uL diluted C0012 1.0 uL diluted C0040 0.54 uL B0034 0.42 uL E0033 3.28 uL pSB1C3
41.5 uL dH2O 42.02 uL dH2O 40.95 uL dH2O 41.5 uL dH2O 41.5 uL dH2O 41.5 uL dH2O 41.96 uL dH2O 42.08 uL dH2O 39.22 uL dH2O
5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4 5 uL NEBuffer 4
0.5 uL BSA 0.5 uL BSA 0.5 uL BSA 0.5 uL BSA 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 EcoRI-HF 1.0 uL XbaI 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF 1.0 uL XbaI 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF
1.0 uL SpeI 1.0 uL SpeI 2.0 uL PstI 1.0 uL SpeI 1.0 uL SpeI 1.0 uL SpeI 2.0 uL PstI 1.0 uL SpeI 2.0 uL PstI

The digestions were mixed by flicking the tube was quick spun to bring liquid to the bottom of the tube. They were incubated in a 37 C water bath for 15 minutes, and then incubated in an 80 C water bath for 20 minutes. Double the amount of PstI enzyme was used because in NEBuffer 4, PstI has a 50% efficiency compared to NEBuffer 2.

Ligations came next:

Ligations
1 2 3 4 5
11 uL dH2O 11 uL dH2O 11 uL dH2O 11 uL dH2O 11 uL dH2O
2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer 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 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase
2.0 uL Digest 2 2.0 uL Digest 3 2.0 uL Digest 1 2.0 uL Digest 3 2.0 uL Digest 3
2.0 uL Digest 3 2.0 uL Digest 4 2.0 uL Digest 7 2.0 uL Digest 5 2.0 uL Digest 8
2.0 uL Digest 9 2.0 uL Digest 9 2.0 uL Digest 9 2.0 uL Digest 9

The tubes each ligation was made in was mixed by flicking and then quick spun down. The ligations were incubated at room temperature for 10 minutes, and then incubated in 80 C for 20 minutes.

Transformations were done next by mixing 50 uL of NEB dH5α competent cells and 3 uL of the ligation liquid to the tubes. I used flicking to mix the cells and ligation. Then this was transfered to a electrocuvette and electroporation was done. After 1 mL of SOC media was added in the electrocuvette and then put in the 37 C room to incubate for an hour. Then the cells were mixed back into the media by pipetting up and down and 200 uL of the transformants were plated one Chloramphenicol plates. Those were put into the 37 C room to overnight.



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