Resuspension of all acquired DNA parts and primers. Made working concentration of primers and amplified two parts (eilRPjexD.5 and PJexD.5-rfpterm) needed to construct our PER1 plasmid containing the EilR repressor and RFP as a reporter gene.
Also attempted to amplify vector backbone from stored pBMTL-2 cell stock.
5/29/24
Elle
Checked results of PCR amplification on gel, eilR and rfp amplification was succesful and bands were acquired, pBMTL-2 vector backbone was not.
Began an overnight culture in order to miniprep the pBMTL-2 from
5/30/24
Elle
Miniprepped the overnight culture of e. coli containing the pBMTL-2 plasmid.
Ran a gel to determine whether or not the plasmid backbone was succesfully isolated, it was not.
5/31/24
Elle and Drake
Used the miniprep for another PCR reaction and increased the extension time, succesfully isolated the backbone via gel confirmation.
Used Dpn1 restriction digest on the backbone, left over the weekend.
6/3/24
Elle
Cleaned the digestion of the pBMTL-2 vector, nanodropped to determine concentration and purity. Results were not satisfactory.
Reamplified the vector from the stored miniprep, as well as the eilR and RFP inserts. Ran on gel to determine succesful isolation/amplificaiton. Gel confirmed that we had desired products.
Dpn1 digest of the pBMTL-2 vector PCR product overnight.
6/4/24
Elle
Cleaned the Dpn1 digestion, nanodropped for concentration and purity. Came back with 8.7 ng/ul. Also measured the concentration of the inserts.
Did calculation and set up Gibson Assembly, transformed Gibson into Dh10b competent cells, and then spread on selection plates.
Drake
Performed PCR reactions to amplify pBMTL-2 vector, eilR and RFP inserts.
Corbin
Cleaned backbone and inserts with MONARCH cleanup kit. Nanodropped all three fragments to find concentrations. Performed Gibson Assembly of the three fragments. After Gibson Assembly, transformed D10 cells and plated for overnight growth.
Components
Volume
Forward Primer
1.25uL
6/5/23
Elle
Set up a colony PCR for determination of succcesful plasmid incorporation into competent cells.
Checked PCR products on gel.
Gibson Assembly and transformation did not work, as shown in the below image.
6/6/23
Elle
Fill in here
Corbin
6/7/23
Elle
Fill in here
Corbin
6/8/23
Elle
Fill in here
6/10/23
Elle
Fill in here
Corbin
Drake
Created Gibson Assembly with pBMTL-2 vector, eiLR and RFP inserts, and conducted transformation protocol to insert plasmid into DH10B competent E. coli cells.
