Miniprep and determine concentrations

From 2007.igem.org

Latest revision as of 03:07, 27 October 2007

Miniprep Protocol

Calculate the approx. S/N ratio of the transformation.

Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise.

Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available.

Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.)

Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube.

Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try.

Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.

Miniprep DNA Extraction

Transfer the overnight culture of plasmid cells into 2ml microcentrifuge collection tubes (1 per try)provided in the kit. Pellet for 1 min. Decant all the liquid and add 1 ml of the culture into the corresponding tube.

Make sure not to mix up the tries.

Add 400ul of ice cold lysis buffer (make sure enzyme mix is already added. Lysis buffer is stored at 4C) Vortex for exactly 30 sec. Let it stay at RT for 3 mins.

Setup filters and collection tubes.

Transfer the contents to the filter + collection tube setup, each try in a separate filter.

Centrifuge for 1 min.

Add 400 µl of Eppendorf Fast plasmid wash buffer. (make sure isopropanol is already added). Spin for 1 min. You need not dicard the contents before this step.

Remove the Spin filter from the tube, discard the filtrate and put the tube back. Spin for 1 min to get rid of the residual alcohol.

Transfer the filters to a new clean and autoclaved 2ml eppendorf tube.

Label the tubes. Add 50 µl of EB per column and wait 3 min.

Spin for 5 mins.

Measure the concentration of DNA.

Digest the tries which seem to have the correct supercoil size with an appropriate restriction enzyme to verify plasmid construction. Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme. You need atleast 100ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 1 µg as they will be divided as 900ng of the 4.5kb band and 100ng of the 500 bp band.

Run on the agarose gel for as long as required to obtain maximum resolution.