Restriction Enzymes and Plasmids

review notes and lab handouts

restriction enzymes

enzymes that cut DNA in certain spots

restriction sites

the specific spots where restriction enzymes cut DNA

How can you tell if a DNA sequence is a restriction site?

the sequence must be a palindrome in order for it to be a restriction site

purpose of using restriction enzymes in biotech

helps to isolate gene of interest and insert it into a plasmid that replicates

types of restriction enzyme cuts

blunt ends and sticky ends

difference between blunt and sticky ends

blunt ends are cut right through (only phosphodiester bonds); sticky ends leave a tail (cuts through phosphodiester and hydrogen bonds)

Why are sticky ends better to use when inserting genes?

blunt ends could be put together backwards

plasmid

DNA that replicates independently and contain genes for unique stressful situations

plasmid maps

shows location of major landmarks on the DNA

ori

origin of replication

polylinker

aka multiple cloning site; contains restriction sites and where the GOI would be inserted

antibioitic resistance

genes that resist antibiotics like ampR, the resistance gene for ampicillin

lacZ

area used to determine if the GOI was inserted correctly

how to place gene into plasmid

1. cut GOI and plasmid with the same restriction enzyme (try to cut as close to GOI as possible)

2. leave sticky ends that will adhere to each other with hydrogen bonds

3. DNA ligase creates phosphodiester bonds between the nucleotides

4. recombinant DNA is inserted back into bacterium

transformation

the uptake of DNA from the environment (such as electro-poration)