Team:UCC Ireland/Project/Notebook/10/07
Monday-10/07/17
Mini digest of plasmids using EcoR1 and Pst1:
- BBa_K1357008 (purple)
- BBa_K1357009 (amilCP)
- BBa_l13600 (cfp)
Ran these samples on gel
Correct bands obtained.
3A Assembly protocol begun - Desired results:
- Purple+cfp
- AmilCP+purple
- Negative control (just backbone)
Began by carrying out a restriction digest on the plasmids using the 3A assembly
Transformation of:
- BBa_K592009 (AmilCP blue)
- BBa_E1010 (AmilCP red)
- BBa_K592011 (AmilCP green)
- BBa_K145279 (GFP TetR)
- BBa_K592012 (AmilCP pink)
Tuesday-11/07/17
No coloured colonies on plates yet.
3A Assembly continued:
Ran a gel in order to check that plasmids had been correctly cut for our 3A Assembly protocol.
Marker, Backbone, Backbone, Backbone, AmilCP+EcoR1+Spe1, Purple+Xbal+Pst1, Cfp+EcoR1+Spe1, Purple+Xbal+Pst1.
Correct bands obtained.
Performed a ligation reaction and transformed the ligated plasmid into E.Coli.
Wednesday-12/07/17
Plasmid preps of AmilCP plasmids (coloured plasmids).
3A assembly results:
- Negative control grew
- One/Two colonies grew in 3A assembly plates
- Enzymes did cut as shown from gel.
Meeting with IGNITE - Business plan
Meeting with Dr. Paul Young -2pm
- Need to clone promoter genes using 3A assembly with reporter genes (AmilCP)
- GFP+TetR plasmid - test with concentrations of tetracycline (BBa_K145279
- BBa_K577895 (TetR and RFP plasmid)
Thursday-13/07/17
- Plasmid preparation of ligated plasmid (cap reported+purple reported+amp backbone) from overnight liquid culture
- Restriction digest on the following plasmids to check correct plasmids are obtained. (This test is needed as colonies did not produce any colour on plate. No promoter was present in plasmid to produce colour):
- 1. Ligated Amp. backbone + BBa_I13600 (cap reporter) + BBa_K1357009 (purple reporter)
- 2. BBa_K592009 (AmilCP blue)
- 3. BBa_E1010 (AmilCP red)
- 4. BBa_K592011 (AmilCP green)
- 5. BBa_K52012 (AmilCP pink)
- Ran a gel with these samples - both uncut and cut.
