IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/08/05

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August 5, 2010

We began the day with a electrophoresis gel of the digestions from 7/29/10 to see if they worked since the transformations didn't work.

We figured out that the backbone I used was not the pSB1C3 with RFP in it that we wanted to use. The digestion also showed that maybe the cells we mini-prepped from had some other plasmid in them at ~6kB.

After finding this we started digestion of the pSB1C3 backbone that has the RFP so we could try digestion/ligation/transformation again with our genes from 7/29/10.

  • Digestion of pSB1C3:
    • 1.1 uL pSB1C3 backbone
    • 41.4 uL dH2O
    • 5.0 uL NEBuffer 4
    • 0.5 uL BSA
    • 1.0 uL EcoRI-HF
    • 1.0 uL PstI

I flicked the solution to mix, then did a flash spin to bring the liquid to the bottom of the PCR tube. I did 1 hour 37 C bath to let the enzymes work, then inactivated them with a 20 minute 80 C bath.

Ligations came next:

Ligations
A B C D E
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 pSB1C3 2.0 uL Digest pSB1C3 2.0 uL Digest pSB1C3 2.0 uL Digest pSB1C3 2.0 uL Digest pSB1C3


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 75 uL of our home made 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.

Francis also did some digestion/ligation/transformations of his own:

Digestions
1 2 3 4
10.0 uL Hfq 0.75 uL pSB1C3 10.0 uL GadX 20.0 uL GadY
32.50 uL dH2O 41.75 uL dH2O 32.50 uL dH2O 22.50 uL dH2O
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
1.0 uL EcoRI-HF 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF 1.0 uL EcoRI-HF
1.0 uL SpeI 1.0 uL PstI 1.0 uL PstI 1.0 uL PstI

He incubated in a 37 C water bath for 30 minutes. He then put the samples into the 80 C bath for 20 minutes.

He then continued with ligations: Ligations came next:

Ligations
A B C
13 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
1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase
2.0 uL Digest 1 2.0 uL Digest 2 2.0 uL Digest 2
2.0 uL Digest 2 2.0 uL Digest 3 2.0 uL Digest 4

He let them incubate at room temperature for 15 minutes and the put them in the 80 C bath for 20 minutes. He then mixed 50 uL NEB turbo cells with 3 uL of ligation buffer in a electrocuvette and electroporated the cells. He then added 1 mL SOC broth and put them into the 37 C room for 1 hour. After this he plated on CAP plates and put in the 37 C for overnight.


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