AAV DNA Bacterial Growth

Transformation

methodology

A bacterial transformation is the process of a competent bacteria cell taking up foreign DNA. The bacteria’s cellular machinery will then replicate this DNA to produce enough for future manipulation and analysis.

The DNA used in transformations is typically in the form of a plasmid which is circular double stranded DNA that is outside of the chromosomes. This DNA will contain an antibiotic resistance gene that the bacteria will express. The corresponding antibiotic is used in the transformation and growth process to prevent the growth of any other bacteria.

Competent bacteria cells are bacteria cells (usually e.coli) that have been treated to easily absorb DNA.

protocol

– Combine 3-5ul of competent cells with no less that 10ng of DNA.

We strongly prefer mach1 competent cells.

– Incubate on ice for 30 minutes.

– Heat shock at 42 degrees celsius for 45 seconds.

– Incubate on ice for 2 minutes.

– Add 150uL of S.O.C. media or LB media to the competent cells.

– Add directly to LB/ampicillin agar plate and spread with beads. For ligated or tough to grow plasmids incubate at 225rpm and 37℃ for 1hr before adding to the plate and spreading.

– Place streaked agar plate in incubator at 37℃ overnight (~16hrs).

Stab Streaking

methodology

Agar stabs are similar to agar plates in that they contain agar made of luria broth however, the agar is put into a glass vial rather than a plate and the bacteria is stabbed into the agar instead of spread across the top.

This is a common way providers will ship plasmids and often it is recommended that the stab is used within two weeks. We recommend streaking from the stab within a week of arrival as bacterial growths from stabs have a lower success rate than streaks made from transformations.

protocol

• Flame your inoculating loop until it gets red hot.
• Allow loop to cool to room temperature.
  • Tapping it on the agar at the edge of the plate can help speed this up.
• Push the inoculating loop into the stab’s puncture and swipe the loop around the upper rim of the puncture to maximize bacteria recovered.
• Then streak the plate by lightly laying the loop on the agar and running it across a section of the agar as shown in the diagram below.
• It is also useful to do multiple streaks per stab in other sections of the plate so if you have too much bacteria on the loop to produce a single colony, the other sections of streaks should have less as you continue to swipe the loop across the agar.
• Place streaked agar plate in incubator at 37℃ overnight (~16hrs).

Amplification

methodology

We amplify DNA using bacterial growths. Bacteria proliferate via binary fission in a nutritional media that is treated with an antibiotic that corresponds to the antibiotic resistance gene being expressed by the bacteria of interest.

We first do an 8 hour starter growth where the bacteria is incubated in a smaller amount of broth to allow the bacteria to leave the lag phase of growth and enter the log phase before being inoculated into a larger growth to later be lysed and cleaned to obtain the extra chromosomal DNA of interest.

• Retrieve the agar plate and check to see if a single colony can be identified. You want to get a single spot of growth that isn’t touching any others and is of average size for the plate so groups, smudges, very large, or very small spots of growth should be avoided
  • If you do not see a single colony because there is too much growth on the plate we suggest redoing the streaking the following afternoon and using less of the transformation than the previous time (may be helpful to plate multiple amounts to increase the chance of one working)
  • If you do not see anything on the plate at all you can leave the plate at 37degC until the afternoon but be aware this introduces more risk to the plasmid. If there is still no colonies and you are confident that your transformation was done correctly then redo the transformation and lower the ampicillin concentration in the agar plate by half (this is rarely necessary but it may be worth trying).
• Prepare a 14mL round bottom tube with 3-5mL of LB broth and ampicillin at a 1:1000 ratio (3mL LB would need 3uL ampicillin)
• Now take a sterile toothpick or pipet tip and lightly scrape the tip over the colony. You don’t need to dig into the gel just roll it over, even if you can’t see bacteria on the tip there should be some picked up.
• Drop your tip into the LB/ampicillin and let that incubate all day (~8hrs) at 37℃ and 225rpm. Be sure that the tubes are secured into the incubator before shaking as it will create more movement of the broth and increase growth.
• Prepare a conical flask with LB and ampicillin at the same concentration as the starter growth (1:1000). The amount of LB/AMP that you use will depend on the size of growth you want to do. For a 250-300mL growth (a typical midi prep) we would use a 1L conical flask.
  • Try to not to put too much LB in one flask as it will lower the amount of movement inside the flask and lower the growth efficiency.
• At the end of the 8 hour 3-5mL starter growth incubation, use the entire starter growth to inoculate the large 250mL growth and secure into a shaking incubator overnight (16 hours) at 37℃ and 225rpm.
• After the overnight large growth we take the OD600 of the large growth and then aliquot 1.5mL of the growth to mini prep with a ZymoPURE plasmid mini prep kit (VWR, 77001-480) before pelleting the large growth and holding the bacterial pellet at -20℃ until the quality control (QC) can be completed.
• We use the purified DNA from the mini preps to QC the plasmids using full length sequencing at Plasmidsaurus.
  • Plasmidsaurus provides full length sequencing allowing us to check the structure of our plasmids and purity of DNA preps. Please check our AAV DNA QC guide for more information on how we judge our Plasmidsaurus results
• Once our plasmid sequences are verified, we purify the DNA using the Macherey Nagel NucleoBond Xtra Midi kit for transfection-grade plasmid DNA (Macherey Nagel, 740410.50).
  • We follow the kit instructions closely, however it is important to remember that the amount of each purification buffer you use in the kit should be adjusted to the amount of bacteria in the prep as measured in the OD600.

Page Written by Aaliyah O’Dell, Research Technician