Team:UCC Ireland/Project/Notebook/17/07
Monday-17/07/17
Plasmid preps of:
- BBa_K592012 (AmilCP pink)
- BBa_K592011 (AmilCP green)
- BBa_K1357009 (amilCP blue)
- BBa_E1010 (AmilCP red)
- BBa_J23119 (Promoter)
3A assembly protocol began:
- Restriction digest of promoter, AmilCP pink, AmilCP blue.
- Ligation of promoter + gene + backbone.
Transformation of plasmids containing promoters:
- BBa_I14033 - P(Cat)
- BBa_J23100 - Constitutive promoter
- BBa_J23101 - Constitutive promoter
- BBa_J23117 - Constitutive promoter
Tuesday-18/07/17
Plasmid preps of the following promoters:
- BBa_J23119 x2
- BBa_K592012 (AmilCP pink)
- BBa_K592011 (AmilCP green)
- BBa_K1357009 (amilCP blue)
- BBa_E1010 (AmilCP red)
Used the nanodrop to quantify the purity and amount of DNA in the samples
Chose BBa_592012 and BBa_E1010 to digest along with the promoter.
Ligation reaction of BBa_592012 with the promoter in the Amp backbone
Ligation reaction of the BBa_E1010 with the promoter in the Amp backbone
Transformation of the ligated plasmids onto Amp plates.
Fom previous day: 3A assembly continued:
- Transformation of the frozen ligated plasmids, and plated onto Amp plates.
Colonies present on pCat plate: Overnights made from these cultures.
0.8% w/v agarose gel ran of uncut BBa_J23119 vs cut BBa_J23119. Against a 1 kb ladder. 5µl of the ladder was loaded into the first well and 15µl of each sample of the 2nd and third wells.
- Plasmid DNA was retrieved from the distribution kit and mini-prepped. The concentration of the plasmid DNA was checked using a nano-drop apparatus and was detected as 22ng/µl of nucleic acids.
- A restriction digest was carried out using Ecor1 and Pst1 to cut the plasmid seen below into fragments of 77BP and 2,028BP, which can be seen visualised on the gel to the right versus the uncut plasmid.
Wednesday-19/07/17
Colonies on 3A assembly plates except the colonies are colourless!!?? - No ribosome binding site.
Overnights made from the colourless colonies - To be tested on a gel.
Plasmid preps of pCat promoter and tested on nanodrop. Restriction digest on the sample.
Transformation of the following:
- BBa_J06702 - mCherry, Plate 3, 15B
- BBa_K592025, Plate 1, 19C
- BBa_I13502
Thursday-20/07/17
Overnights made of the transformed colonies from Wednesday.
Plasmid prep of E1010 and K592012, nanodrop analysis;
- E1010 - 47ng of Nucleic acids/ml
- K592012 - 48ng of Nucleic acids/ml
- K592012 - 45.7ng of Nucleic acids/ml
- K592009 - 46.2ng of Nucleic acids/ml
Cut all of the plasmid preps using Ecor1 and Pst1 and ran on a 0.8% agarose gel to check that the correct recombinant plasmid had been taken up by the competent cells. Predicted fragment sizes are as follows:
- E1010 - 741BP and 2,155BP
- K592012 - 716BP and 2,155BP
- K592009 - 704BP and 2,155BP
Strange results obtained. Possible reasons for fragments of approximately 2000bp seen:
One of the restriction enzymes, EcoR1 or PstI, did not cut the ligated plasmid since the restriction sites might have been destroyed during ligation
Friday-21/07/17
Plasmid prep of yesterday’s overnights: BBa_J06702 - mCherry, BBa_K592025 and BBa_I13502. Digested these plasmids with Xba and Pst.
Promoters (PCat and J23119) digested with EcoR1 and Spe.
Backbones (tet) digested with EcoR1 and Pst1.
Met Yensi, shown how to use BMG fluostar omega plate reader in CCRC.
Ligation:
Equation: ng insert = vector ng x (kb size insert/kb size vector) x molar ratio (3) Carried out a ligation reaction.
Made overnights of TetR, GFP-TetR, Promoter-RFP, Promoter-GFP.
