Restriction digestion DNA concentration Gel electrophoresis

DNA gel electrophoresis When running DNA gels with ethidium bromide the intesity of the band will reflect the amount of DNA Thus when digesting a DNA sample short fragments will be less intense than longer fragments Supercoiled and open circular DNA molecules (relaxed plasmids with a nick in one of the strands in the DNA double helix) move differently in the gel compared to linear fragments The percentage of agarose can be optimized for identification of shorter or longer fragments Normal yield for a plasmid miniprep 300-400 ng/µl undigested linearized Nicked Super- coiled

Measuring DNA We will be using a Nanodrop instruent to measure DNA concentrations. Use 1 ml of your plasmid prep. A solution containing 50 µg/ml of double stranded DNA has an absorbency (optical density) of 1.0 at a wavelength of 260 nm. 50 µg/ml /1.0 = [DNA]/OD read [DNA] = 50 µg/ml x OD read DNA mg/ml = ODA260read x 50 µg/ml. If you have diliuted the DNA you have to multiply with the dilusion factor A260/230 should be around 1.8 for pure DNA and around 2 for pure RNA

Measuring DNA DNA quality measurement is based on the fact that OD at 260 nm is twice that at 280 nm and 230 if the solution contains pure DNA. If there is a contaminant, OD ratio decreases. Clean DNA has a ODA260 /ODA280 and a ODA260 /ODA230 between 1.8 and 2.0, if value is smaller it indicates a contamination.

pCR 2.1-TOPO 909 763 Primers for sequencing: M13F and M13R

Restriction of clones SUVR1 NcoI (4601) EcoRI (4414) DdeI (4240) NcoI (4134) 467+ 2548+ 1679

Restriction of clones SUVR1 RC 467+ 2436 + 1791

Restriction of clones SUVR2

Restriction of clones SUVR2 RC

Restriction of clones SUVR4

Restriction of clones SUVR4 RC

Restriction of clones ASHH2

Restriction of clones ASHH2 RC

Restriction of clones - ASHR3 ASHR3 RC 4866 bp (4825) (4660) (4595)

Restriction of clones - ASHR3 RC (3963) (4169) (4234) 4866 bp ASHR3 RC

Which restriction enzyme to use? Different enzymes require different buffers When making double digests you might have to compromise on the buffer you use so it is optimized for both enzymes, this may require using a buffer that does not provide 100% activity for both enzymes. In your lab journal you should write the expected fragment sizes and in which orientation your gene is cloned based on the results you get.

Restriction of clones SUVR2 G

Restriction of clones SUVR2 G RC