Bacterial transformation

if you remember the workflow of cloning so the first step is to digest your vector or your plasmid with restriction enzyme and also digesting your gene of interest with the same restriction enzyme and thereby we perform a ligation reaction which would put our gene of interest inside the plasmid inside the vehicle or inside the vector and then we would perform a transformation reaction the transformation would allow the plasmid to be incorporated inside a compatible bacteria which is able to like take up the plasmid and allow the plasmid to be expressed and then we grow the bacteria in large amount in cultures so in this video I will be talking about the transformation process and how it is actually done and what is the rationale behind it so simply transformation of plasmid means putting a plasmid which may contain your gene of interest inside a bacteria so the process is fairly simple so the way we do transformation is by creating small pores in the cell wall and the cell membrane by intubating it with divalent cation and a hit shop so which would create transient pore in the membrane and that would allow the plasmid to be incorporated inside the bacteria and later on if you give enough time for recovery for the bacteria it would kind of repair their damages and it would grow in the media nicely and it would also amplify the number of copies of our gene of interest which is inside the plasmid so let's just look at the process step by step so the first step of heat shock method transformation is taking the plasmid which has your gene of interest and adding that to a new tube then we add competent cell most of the cases the competent cell lines are t h5 alpha and there are many other components cell lines which are capable of taking the or incorporating the plasmid now after that we incubate the plasmid and the component cell mixture for several minutes once it is chilled on ice we quickly put it into a water bath which is in 42 degrees centigrade and keep it strictly for 90 seconds then we take it out and put it back into the ice so we have the temperature changing from 4 degree to 42 degree centigrade for brief time point and then again back to 4 degree centigrade so this would create a heat shock and after two minutes of incubating in the ice what would happen is there would be transient pour in the bacterial cell membrane and the cell wall and that would allow the plasmid to get inside the bacterial cell then the now the bacterias are in stress so they need to grow and that's why we take the transform and solution and put it into a so same media now SOC media is basically a media which is like enriched in tryptone and a lot of glucose so this media would allow the bacteria to grow because if we indirectly plate it into the plate so the plate already has some kind of raised antibiotic resistance right now the in order for the and rub antibiotic resistance to work the bacteria need to give we need to give the bacteria enough time for these antibiotic resistance protein to be formed right so we have to incubate the bacteria for 30 minutes or so so this would give the bacteria sufficient time to produce the product of the antibiotic resistance gene now what we would do is put the bacteria or played the bacteria into 10 cm in 10 centimeter plate with a desired antibiotic resistance let's say ampicillin resistance so we would put ampicillin in the plate and the bacteria should have the ampicillin right then we would inoculate the colonies and spread the colonies all over the plate and if we wait overnight and then check the there should be colonies in the plate and ideally this colony should be the Transformers right but in order to check it we should take out the colonies and we can also calculate the transformation efficiency by the following formula that is shown in the screen right now but we have to like pick the colony out and then grow it in large cultures and then it could be used for downstream processes like plasmid isolation and thereby followed by sequencing to understand whether the cloning has worked properly or not and so on so forth so this is one of the most important technique done in molecular biology labs and I hope you enjoyed this video if you like this video give it a click thumbs up don't forget to Like share and subscribe thank you

Channel: Animated biology With arpan

Views: 6,981

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Keywords: bacterial transformation, transforming a bactera, transformation, gene cloning, molecular cloning, iit jam biotechnology, csir ugc net, biological sciences

Id: h9BFBJJl3pg

Length: 5min 21sec (321 seconds)

Published: Wed Jul 31 2019