I have a problem with your use of two different restriction enzymes to cut the palsmid and gene of interest, won't this affect the joining of sticky ends through hydrogen bonding before they are ligated?
Using the two different enzymes allows for directional cloning. If you cut both ends with the same enzyme the insert has a 50% chance of going in backwards. The sticky ends will still work at each end with two different enzymes as long as the plasmid and insert use the right enzymes, but won’t work if the insert goes backwards as they won’t have the right hydrogen bonds (as you identify) - this gives you more control over your cloning. Hope that helps.
Not usually - restriction sites are very specific so any variation in sequence will stop the site being recognised - this can be exploited in genetic engineering as if you want to add/remove a site you only need to modify a small number of base pairs
Another great video! Thank you!
Thank you for all your support! Great video !!!
Great video !!
really nice ,
thanks
I have a problem with your use of two different restriction enzymes to cut the palsmid and gene of interest, won't this affect the joining of sticky ends through hydrogen bonding before they are ligated?
Using the two different enzymes allows for directional cloning. If you cut both ends with the same enzyme the insert has a 50% chance of going in backwards. The sticky ends will still work at each end with two different enzymes as long as the plasmid and insert use the right enzymes, but won’t work if the insert goes backwards as they won’t have the right hydrogen bonds (as you identify) - this gives you more control over your cloning. Hope that helps.
Hi is there wobble in the recognition of the restriction site
Not usually - restriction sites are very specific so any variation in sequence will stop the site being recognised - this can be exploited in genetic engineering as if you want to add/remove a site you only need to modify a small number of base pairs
Another great video! Thank you!