By Basset A.B.
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As a general rule, to set up a restriction enzyme digest, determine the amount of DNA to be cleaved, use a fivefold excess of enzyme, ensure that the volume of enzyme does not exceed 10–20% of the final volume, and add 10× buffer to a final concentration of 1×. 5 µg/µl, the following reagents would be added, in order: DNA Water 10× buffer Enzyme (10 U/µl) 4 µl 13 µl 2 µl 1 µl Total volume 20 µl The reaction could also be set up with 3 µl of water and 1 µl of buffer for a total volume of 10 µl. ” For this reason, the enzyme should be diluted at least 1:10 and should be added last, to the diluted enzyme buffer.
To orient you, here is a summary of the next series of experiments: G 1. Cut vector pET-41a(+) (today). 2. Perform restriction digest of pEGFP-N1 and run preparative gel to isolate egfp fragment. 3. Ligate insert (egfp) to vector (pET-41a(+)). 4. Transform ligation products into competent cells. 5. Pick putative transformants and make replica plates. 6. Identify positive clones. I. INTRODUCTION In general, cloning vectors are plasmids that are used primarily to propagate DNA. They replicate in E.
3. Using sterile technique and a sterile 5-ml pipette, withdraw samples from the cultures and determine the OD600 using a spectrophotometer (Spectronic 20 or equivalent). Once cultures appear cloudy, they are in log phase growth and their OD will increase rapidly. 4. When the proper optical density is reached, pour the culture into two sterile Oakridge tubes. Balance the tubes and then place on ice for 15 min. From now on, it is important that the cells never warm to room temperature. 5. Harvest the cells by centrifugation at 6000 rpm for 10 min at 4˚C.