Analysis of transformants

2.8.1 Plasmid purification (mini-preps)

QIAprep® Spin Miniprep Kit (Qiagen) or GeneJET™ mini-prep kit (Thermo- Fermentas) were used for purification of plasmid DNA from small volumes of liquid culture. Both kits follow a similar protocol involving alkaline lysis of bacterial cells and binding of DNA to silica membrane columns. Prior to plasmid DNA purification, liquid cultures of E. coli containing plasmid DNA were set up in 15 ml Falcon tubes containing 4ml of low salt LB broth and 50 µg/ml of appropriate antibiotic and incubated for at least 18 hours in a shaking incubator at 250rpm, 30- 37oC.

Cells were harvested by centrifugation at 4000g for 15 minutes at 10oC, the supernatants removed and the pellets re-suspended (by pipetting and vortexing) in 250µl re-suspension buffer. Cell suspensions were transferred to sterile 1.5ml eppendorf tubes and cell lysis was performed by adding 250µl of lysis buffer to each tube followed by gentle mixing until a homogenous solution was obtained. Each lysis reaction was terminated by adding 350µl of Neutralisation solution. The mixtures were then centrifuged for 8 minutes at 10,000g at room temperature to pellet the cell debris and denatured proteins. Supernatants were then transferred to DNA binding columns. A brief 1 minute centrifugation at 10,000g allowed DNA to bind to the column resin which was then washed once with 700µl of wash buffer (QIAGEN) or twice with 500µl of wash buffer (Thermo-Fermentas). Columns were then spun for a further 1 minute at max speed to remove any residual wash buffer. DNA was eluted from the columns with 50µl of EB buffer. The quality of the DNA was checked by A260/A280 measurements using a NanoDrop1000 (Thermo Scientific) spectrophotometer. For inserts over 8Kb analytical agarose gel electrophoresis was used to check the recovered DNA (see 2.5.4).

44 2.8.2 Restriction digests

All plasmids containing inserts larger than 8Kb were checked for possible rearrangements using restriction enzyme digests. Expected restriction fragment sizes were predicted in silico using Vector NTI v10.5 (Life Technologies) or Geneious v.6.6.5 (Biomatters Ltd., Auckland, NZ) software. Typical diagnostic BglII or SalI (Promega, WI, USA) digests were set up in a 20µl reaction as follows: 2µl 10x buffer D (Promega), 2µl 10x BSA, 1µl BglII or SalI (10U/µl), 1µg of plasmid DNA, nuclease free water up to 20µl. Reactions were briefly vortexed and then incubated in a PCR block at 37oC for at least 1 hour, 15 minutes. Products were run on 0.8-1% w/v agarose TAE electrophoresis gels for visualization and analysis.

2.8.3 Sequencing

Sequencing done in-house at Rothamsted utilized the Big Dye Terminator v1.1 kit (Applied Biosystems®-Life Technologies, CA, USA) and an ABI 3100 genetic analyser (Hitachi, Tokyo, Japan). A typical 20µl sequencing reaction was set up as follows: 4µl Big Dye v1.1 reaction mix, 2µl 5x BigDye Sequencing buffer, 1µl sequencing primer (20pmol), 500ng Plasmid DNA (or 100ng purified PCR product), nuclease-free water up to 20µl. The PCR block cycle conditions were 25 cycles of: 96oC for 30 seconds, 50oC for 30 seconds 60oC for 4 minutes with the temperature ramp set at 1oC per second.

Dye labelled DNA was precipitated from the reaction mix by the addition of 2µl of 125mM EDTA, 2µl of 3M NaOAc (pH 5.5) and 71µl of absolute ethanol, brief vortexing, then incubation at room temperature for 15 minutes. The precipitated DNA was pelleted by 20 minutes centrifugation at 10,000g, the supernatant removed and the pellet washed with 100µl of 70% ethanol. Following another centrifugation at 10,000g for 5 minutes, as much of the supernatant as possible was decanted by inverting the tube, and the DNA pellet was allowed to air dry in the dark until all the residual ethanol had evaporated. The DNA was then re-suspended in 20µl of Hi- Di™ formamide (Life technologies) and transferred into an individual well on a 96 well plate. Prior to sequencing all reactions were briefly incubated at 90oC for 2 minutes.

Chapter II

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In some cases sequencing was done commercially by MWG ERUOFINS DNA (Eurofins Scientific group, Brussels, Belgium).

2.8.4 Maxi preps

2.8.4.1 Liquid cultures

A colony of E. coli with desired plasmid was inoculated into a 2.5ml starter culture of LB broth (containing 50µg/ml of antibiotic) and propagated in a shaking incubator at 30oC, 250rpm. After approximately 8-10 hours, 1ml of the starter culture was used to inoculate two 200ml LB broth cultures (containing 50µg/ml of antibiotic) and incubated with shaking overnight at 30oC, 250rpm. Before harvesting the OD of the culture at 600nm was checked and if the absorbance value was approximately 1.0 the cells were transferred to 250ml Beckman centrifuge bottles and spun down at 6000g, 4oC for 15 minutes in a JA-14 rotor / Avanti 28 Centrifuge (Beckman, CA, USA). 2.8.4.2 DNA purification

The pelleted E. coli cells were processed with a Plasmid Plus maxi kit (Qiagen, Hilden, Germany) using a modification of the manufacturer’s protocol. Pellets were re-suspended in 8ml of Qiagen kit resuspension buffer P1, containing lysate-blue and RNase A. Cells were lysed by adding 8ml of lysis buffer P2 and gently mixing the solution until the colour of the lysate turned blue. 8ml of neutralization buffer S3 was then added to stop the lysis reaction and the contents of the tube gently mixed until all the blue colour disappeared. The mixture was then centrifuged at 4500g and 10oC for 5 minutes and the supernatant from two 200ml cultures was transferred into one QIAfilter cartridge. The lysate was gently pushed through the cartridge filter (using the plunger provided) and collected in a fresh sterile 50ml tube. 10ml of binding buffer was added to the filtrate, the solution mixed, and the entire contents of the tube applied to a DNA binding column placed on a vacuum manifold. After all the liquid had passed through the column, it was washed with 0.7ml of ETR buffer to remove endotoxins, centrifuged for 1 minute at 10,000g and then washed with 0.7ml of buffer PE and re-centrifuged for 1 minute at 10,000g. The column was then spun again at max speed for 1 minute to remove any residual wash buffer. DNA was eluted from columns using 400µl of EB buffer and a 2 minutes centrifugation at

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8,000g. The quality and quantity of the DNA was checked by NanoDrop1000 spectrophotometry and by restriction digests (see 2.8.1 and 2.8.2 for details).