Beware that some restriction sites might be present in the sequence but “masked” by methylation in the bacteria. Much more on it here: bit.ly/reasesvsmtases but basically bacteria have restriction enzymes as part of a 2-part defense mechanism, the Restriction-Modification system. They restrict viruses from replicating inside them by cutting the viral DNA with restriction enzymes. And to keep from cutting their own DNA, they modify vulnerable would-be cut sites in their own DNA with methylation. Different bacteria have different methyltransferases, which, when we stick a plasmid in bacteria, will methylate the plasmid too. Therefore, you want to make sure the bacteria you use don’t modify the sites you want to use. Thankfully, Most of the REases we use in the lab don’t overlap with a common methyltransferase, Dam, sites, but ClaI, MboI, and XboI might. Another one is Dcm, which can interfere with cutting by ApaI, BsaI, and McsI. And EcoKI can hide cut sites for DraI, HpaI, and PmeI A couple practical notes - The enzymes are “numbered” not “lettered” (e.g. EcoRV isn’t an all-electric RV model, it’s EcoR FIVE (learned this the embarrassing way). It tells you it was the 5th restriction enzyme found in the “RY13” strain of E. coli) - To help you compare, run some controls: negative control: don’t add enzyme - shows you what uncut looks like. also run plasmid-only (you know it doesn’t have insert) and insert-only (you know it doesn’t have plasmid) - You have to purify the DNA first - & you don’t want to waste all your DNA (if you do decide to “hire it” you’ll need the rest - so you typically just test a tiny bit in ~20μL total (1 μL (microliter) is 1 millionth of a liter) We can also we can also use a similar PCR-based method called colony PCR, where we use short pieces of DNA called primers to bookend regions of the plasmid we want copied and then make copies and see how big the copied bits are. Depending on how you design the primers you’ll only get a product (copied bit) if the insert was in there (if you use an insert-specific primer as one of the bookends) or you’ll get different sized products if the insert is or isn’t in there (if you use only vector-specific primers). bit.ly/colonypcretc; bit.ly/colonyPCRchat ; video: kzitem.info/news/bejne/t5tqr217hXSdmHY A nice thing about colony is you don’t have to do the mini prep first - you can work straight from the colony. Which especially helpful if you are trying to screen a number of them. Here’s more on how it works: Speaking of screening - methods like blue-white screening can help you triage which clones to check. Basically, you do your cloning so that your insert is inserted into a gene the bacteria need in order to make a blue product. If your insert (or at least something) gets in there, it disrupts that gene, so the cells can’t make the blue product and those colonies look like normal colonies, not blue. This doesn’t tell you about the size of the thing that got in, so it tells you less than the PCR and restriction cloning, but it can be used to choose which colonies to test. more on blue-white screening: blog: bit.ly/bluewhitescreening ; video: kzitem.info/news/bejne/mouitmZrkoiHf3o But all of these techniques are just helping you choose which are probably okay and then you have to take the probably okay ones and send them for sequencing to be sure all’s spelled okay. These days, sequencing has gotten really cheap and fast so you might just go directly to that for routine cloning. more on clone-checking: bit.ly/colonychecking more on DNA sequencing: bit.ly/DNAsequencingmethods & bit.ly/sequenceclones & bit.ly/sequencetermstools more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
@HaraChoi0153
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Very helpful video, thank you!
@EchoesOfAffirmations
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What could be the reason of having three bands appear in my analytical digest?
@thebumblingbiochemist
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If your plasmid has 2 cut sites you will get 3 bands
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