Hi there! (am using Whatsapp) :D
When I started writing this, I wanted to start with a "Hi" and suddenly, the famous Whatsapp status came into my mind! :P See, am really addicted to Whatsapp nowadays, changing DP and Status often. Oof, I should stop writing whatsapp status and start writing here. (This is NO ADvertisement for Whatsapp :P)
In the last post, I said that I'm in the USA doing research and now, am back in India after successfully completing the internship! Yaaai, back home, back home! It feels good when you are in home, yes, am happy to be home. But, sometimes, its good to visit places and learn new things, leaving home. Let's come to the purpose of this post now! In this post, am gonna share few things I learnt from my internship.
I was basically cloning different fusion proteins and was doing invitro degradation studies of those cloned proteins using western blots. I've done cloning before, when I was doing my under graduation (UG), but, I think I've grown up now to understand things better! Things once I found very difficult have become easy now! I'll give you an example: During my UG, I found it very difficult to cut out the required DNA band from agarose gel for gel extraction. My hands use to do "Disco dance" when I use to try cutting the gel. (I think, many students suffer from this "shaky hand" problem! Don't worry, you'll be alright soon) But, now, guess what? Am doing it like a BOSS! :D I think, it comes with practice or should I say, "I've grown up!"
Back to the point, when I was trying to clone an insert into a plasmid vector, the first time, it was a failure! I got only the vector without any insert. And, then I did trouble shoot the strategy and found out a correct way of doing it, and, in the next attempt, EUREKA, success! :D
Here are few things you should care while cloning (plasmid vector, E.coli):
1) Always try to use two different restriction enzymes
Using two different restriction enzymes will help in avoiding the self ligation of the vector i.e., when you are restricting the vector with only one restriction enzyme and then purifying it, using it for cloning, there is a possibility for your insert to get in the vector but also there is every possibility for your vector to self ligate!
|Self ligation of vector|
2) Do Gel extraction purification whenever possible after restriction
This is where I did mistake when I was cloning. I restricted my vector with two different enzymes, but, I didn't do gel extraction, instead, I did PCR cleanup! PCR cleanup can generally remove upto fragments of 100bp but not the fragments more than 100bp. My vector after restricting with two enzymes, gave out a fragment which was greater than 100bp. As the PCR cleanup was not efficient to remove this fragment, it got re-ligated when I did ligation with my insert! (See picture) The possibility of the actual vector fragment to self ligate is more than that of the insert.
Obviously, doing, gel extraction and purification after each restriction will give you less amount of DNA, but, trust me, even if you have 1 or 2ng/micro litre concentration of DNA, that would be pure and you'll get a good number of positive colonies at the end.
3) Restrict overnight!
Normally, this won't be necessary if your enzyme quality is good. But, I had problem with one of my restriction enzymes when I did 1 hour incubation and hence I decided to do overnight restriction. The overnight restriction at 37 degree Celsius worked fine and gave good number of positive clones at the end.
If you are doing overnight restriction then, it is better to seal your tubes with parafilm to avoid evaporation of the buffer (There are chances for the buffer to evaporate slowly and thus increasing the buffer concentration). There is also possibility for star activity (non specific restriction due to varying reaction conditions) but, even if there is star activity, you will ultimately have properly restricted product of interest also in the mixture and this product can ligate properly with your vector.
4) For mutations!
I also did point mutations in the insert, and, I hope it is better to do the mutation after getting the clone, i.e., put your insert in the vector, confirm the sequence and then do the point mutation. By this way, if you need to compare both the native and the mutated protein, you need not do two different cloning, instead, you can have a clone of native and from that you can take some amount of plasmid and do point mutation in it using mutation PCR.
I initially had a doubt, "whether it is possible to do PCR of whole plasmid with the insert, around 6Kb, using PCR?" Later, I found the answer as, "Yes, it is possible to do!". I used Pfu DNA polymerase for the PCR. Pfu is superior than normal Taq DNA polymerase for it has proof reading activity also, hence, less error! The primers which I used for this mutation PCR of plasmid was about 40bp in length.
And, it is necessary to do digestion with DpnI after the mutation PCR. The template plasmid what we are using for the PCR is generally the plasmid obtained from dam+ bacteria, .ie., this plasmid is methylated. We should restrict the PCR product with DpnI enzyme which selectively restricts the methylated DNA, providing us only with the mutated product.
Hope, this piece of information helps someone in cloning. Happy cloning :D All the best!