IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/21

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July 21, 2010

With the cloned out parts and parts from the registry, we began testing digestion/ligation/transformation.

RXN # Digest Cutters Concentrations Volume of template
1 Chloramphenicol resistant plasmid EcoRI + PstI 152.4 ng/uL 3.5 uL
2 Kanamycin resistant plasmid EcoRI + PstI 88.6 ng/uL 6 uL
3 Ampicillin resistant plasmid EcoRI + PstI 218 ng/uL 2.3 uL
4 M30109 EcoRI + SpeI 2254.4 ng/uL 0.2 uL
5 R0082 XbaI + PstI 1125.01 ng/uL 0.5 uL
6 B0034 EcoRI + SpeI 928 ng/uL 0.5 uL
7 E0033 XbaI + PstI 1201.5 ng/uL 0.42 uL
8 K091101 EcoRI + SpeI 2726.1 ng/uL 0.2 uL
9 E0430 XbaI + PstI 885.98 ng/uL 0.58 uL
10 MicF XbaI + PstI 16.78 ng/uL 15 uL
11 MicF EcoRI + PstI 48.07 ng/uL 10 uL

Each one was digested with the following procedure. The restriction enzymes and volume of template of each is on the table above:

  • RXN (50 uL)
    • 5 uL NEBuffer 2
    • 0.5 uL BSA
    • 1.0 uL Restriction Enzyme 1
    • 1.0 uL Restriction Enzyme 2
    • Volume of template from table
    • Volume of dH20 that will make the reaction 50 uL overall

After all of the restriction enzyme and other solutions were added to the reactions, they were put in a 37 C bath for 30 minutes. After that, they were put in a 80 C bath for 20 minutes.

Next ligations of all the digestions was done with the following procedure:

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 buffer 2.0 uL 10X buffer 2.0 uL 10X buffer 2.0 uL 10X buffer 2.0 uL 10X buffer
1.0 uL ligase 1.0 uL ligase 1.0 uL ligase 1.0 uL ligase 1.0 uL ligase
2.0 uL Digest 2 2.0 uL Digest 1 2.0 uL Digest 1 2.0 uL Digest 1 2.0 uL Digest 3
2.0 uL Digest 4 2.0 uL Digest 6 2.0 uL Digest 8 2.0 uL Digest 8 2.0 uL Digest 11
2.0 uL Digest 5 2.0 uL Digest 7 2.0 uL Digest 9 2.0 uL Digest 10

Each was incubated for 15 minutes at room temperature (25 C)

Then they were transformed into 50 uL of dH5α cells with 3 uL of ligation solution.

Ligation 1 was inoculated into 200 uL of LB/Kanamycin broth and overnighted in the 37 C room. All the rest of the ligations were inoculated into 200 uL of LB/Chloramphenicol broth and put in the 37 C overnight.