IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/08/09

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August 9, 2010

Today I wanted to retry all of the digestion/ligation/transformations since they didn't work again.

Digestions
1 2 3 4 5 6 7
0.48 uL E1010 1.55 uL B0015 0.50 uL E0020 0.50 uL C0040 0.54 uL B0034 0.50 uL E0033 0.50 uL pSB1C
42.02 uL dH2O 39.95 uL dH2O 42.00 uL dH2O 42.00 uL dH2O 40.96 uL dH2O 42.00 uL dH2O 41.00 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
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 XbaI 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 2.0 uL PstI 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 1 hour, 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 1 2.0 uL Digest 2 2.0 uL Digest 1 (From 7/29 (J23100)) 2.0 uL Digest 5 (From 7/29 (C0012)) 2.0 uL Digest 4
2.0 uL Digest 2 2.0 uL Digest 3 2.0 uL Digest 5 2.0 uL Digest 2 2.0 uL Digest 2
2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7

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.


Then I transformed the ligations with 75 uL of our home-made electro-competent cells and 3 uL of ligation. They were both put directly into the electrocuvette. After electroporation, 1 mL of SOC media was added to the cells and they were put into the 37 C room for 1 hour. After that 200 uL of cells was plated onto CAP plates and put into the 37 C overnight.


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