Team:UNC-Chapel Hill/Protocols

PROTOCOLS

PCR

PCR amplification was performed using a TECHNE 3Prime thermocycler. PCR solutions were composed of 3 uL 10ng/uL template DNA, 1/2 part F. primer, 1/2 part R. primer, 5 parts 5X Phusion HF Buffer, 1/4 part dNTPs (10mM) and 15.5 parts USP H₂O. For all our parts, we utilized the forward primer 5’ ACC GAC TCT CCG TTG CGT TAA G 3’and reverse primer 5’ TAA GGG TCA CTG GGC GAT ATT CG 3’. Both of the primers were determined to have a melting temperature of approximately 59°C according to the OLIGOANYLYZER 3.1 on IDT’s website.

Transformation

E. coli cells were thawed slowly in ice. After reaching a liquid state, 20 μL of cells were pipetted into clean autoclaved 1.7 mL tubes. Next, 2 μL of DNA was added (plasmids from kit or ligation mixtures) and mixed in tubes by gently pipetting up and down. The mixture was then incubated on ice for half an hour. Cells were then heat shocked for 30 seconds at 42°C and then placed on ice again for five minutes. 500 μL of pre-heated 37°C SOC media was added to the cells and placed in a shaker at 37 °C for one hour. The cells were then pipetted onto the LB plates that had the corresponding antibiotic for the vector. Plates were left to grow overnight (16 hours) in an incubator at 37 °C.

Elongated Digestion

Depending on the type of enzymatic cut required, 1 part of 10uM enzyme was added with 1 part of another 10uM enzyme, 2 parts 10X 2.1 NEB Buffer, 1 part CIP if the targeted DNA is the backbone for insertion, and varying parts of DNA and USP H₂O to digest 500 ng of DNA in 20uL of total solution. These solutions were incubated for 2 hours in the 37° incubator before being used for a gel or purified.

DNA Clean and Concentrator Prep

This prep designed by Zymo Research was utilized to purify digests to prevent CIP interference during ligation (as CIP is not fully heat-inactivated) and to purify PCR runs for digestion. For both of these cases, 5x the volume of the solutions intended for purification was added in the form of DNA binding buffer and the total new solution was loaded on Zymo-Spin columns. These columns were then centrifuged at 14,000 x g for 30 seconds and washed with 200 uL of DNA Wash Buffer for 30 seconds at 14,000 x g. The wash step was then repeated and the columns were transferred to clean 1.7 mL tubes. These DNA in this columns was eluted with 15 mL USP H₂O and allowed to rest for five minutes. The tubes were then spun for one minute at 14,000 x g and the columns discarded to give superpure DNA.

Elongated Ligation

All ligation mixtures were 10 μL in total. Each had 1 μL of T4 DNA ligase and T4 DNA ligase 10X buffer. DNA amounts added varied from 0.5 μL to 4 μL in each individual ligation reaction depending on the concentration of the digestion mixture after the DNA clean and concentrate step. USP H₂O was added until a final volume of 10 μL. These ligation reactions were incubated at room temperature overnight before transforming.

Liquid Culture

Using a 200 μL pipet tip, colonies were picked from the plates and then dropped into clean glass tubes that have 5 mL of LB with the appropriate antibiotic. The cultures were then incubated at 37°C and shaken at 250 rpm for 16-24 hours

Glycerol Stock

Into a 1.7 mL tube, 600 μL of liquid culture and 400 μL of 50% glycerol was added. The tube was then stored in a -80 °C freezer.

ZR Plasmid Miniprep Classic

The Zymo Research based kit was used to extract and purify plasmid DNA from liquid cultures. First the DNA from the liquid culture was pelleted into a 1.7 mL tube using a 16,000 x g spin. Then 200 μL of P1 Buffer was added and mixed well to resuspend the pellet. 200 μL of P2 Buffer followed this and was mixed via gentle inversion of the tubes. The total mixture was incubated at room temperature for 1 to 2 minutes. Next, 400 μL of P3 buffer was added and mixed via gentle inversion The mixture was then centrifuged at 14,000 x g for 2 minutes and the supernatant was added to the ZYMO spin column. The mixture was then centrifuged for 30 seconds at 14,000 x g into a collection tube. 200 μL of Endo-Wash Buffer was added and then centrifuged for 30 seconds at 14,000 x g. Then 400 μL of Plasmid Wash Buffer was added and centrifuged for one minute at 14,000 x g. Lastly, the column was placed into a clean 1.7 mL microtube and 30 μL of USP H₂O was added and allowed to incubate for 5 minutes before centrifuging at 14,000 x g for one minute.

Agarose Gel Electrophoresis

The agarose gel was made by dissolving 1 gram of agarose in 100 mL of 1X TAE buffer and microwaving till the agarose was fully in solution. Then 1 μL of ethidium bromide was added to the solution. The solution was then poured into the molding and cooled until it formed a gel (a comb was added immediately afterwards to create the wells). Once the gel was cooled, it was reoriented so that the wells were on the negative side of the apparatus. 1X TAE buffer was added to the external wells so that it slightly overflowed and covered the gel. The gel was then ready for DNA. The DNA in this experimental design were all cut with EcoRI and PstI, combined with DNA loading dye (1 to 5 ratio) and inserted into the gel. The gels were run for one hour at 120 mV, then analyzed under a High Performance UV Transilluminator made by the company UVP.