From my past experience (personal and fellow students), I can say that a graduate student has three phases.
1.Ignorant phase: You are full of theoretical knowledge. You look at every running project in the lab as a possible site for opportunity.
2.Struggle phase: You start to work on your own project, but, somehow you are not able to figure out why the simple PCR is not working.
3.Desperate phase: By now you have seen the world. You have already tried all the advices “heard” (from friends and supervisor) and “read” (online and even visited the least visited shrine called “library” to find those 1960s paper). But, what a bad luck, nothing is working. You even started a new ritual by visiting online molecular biology forums and posting your questions and waiting for an answer. Sorry, people are suggesting more or less the same stuff. Now at this stage, you can do anything to get the single clone (and that too positive). Looking at your desperation may be molecular biology God will shower his grace on you and you might get a clone. That will be a relief!!! Don’t you think so?
Never mind, I was no different than the one described above. The only problem (and also the solution too) with molecular biology is that it is both science and a tool. I found that there is no thumb rule for getting the right clone each time. Every DNA, vector system, user hands etc are different. But, I did find that with a bit of planning, good tools and resources one can increase the chances of getting the right clones (yes! each time). Before you use this resources guide, just remember the Big mantra of molecular biology: if it ain’t broke, don’t fix it (if things are working normal then don’t be tempted to use this guide).
In this InfoMention I consider molecular biology as a tool. I am sharing some useful resources with you all which I myself reliably use in my cloning procedures. Some of these you may already be using, but there are always simple things which we think are too simple but probably they are worth having a read.
1. pDraw software from AcaClone Software:
In order to do successful cloning, you must know before hand at least how different bits and pieces are arranged e.g. you need to see the plasmid details, like which restriction sites it has, orientation of your gene of interest, protein sequence etc.
For that I recommend to use “pDraw software”. This is absolutely FREE, no strings attached. Although, their website is not very attractive (looks like from Windows 95 era), but their software is brilliant. Just hit the “download” button and install it. Downloading and installing is quite self explanatory. I used this software to design my cloning strategy, RE mapping, Agarose gel simulation, Primer designing, etc. And here I can say “it works”.
Web Address: http://www.acaclone.com/
2. Addgene – A Better Way To Share Plasmids:
In research, we often think that if a construct is cumbersome to make, why don’t we just buy it from the commercial companies like the other laboratory chemicals. Well, here is good news for you. You have Addgene as a shot in your arm.
Addgene is a non-profit organization dedicated to making it easier for scientists to share plasmids. Visit their website and you will know what you want to do next. You can search Addgene site using PI name, plasmid name, gene name etc. You need to make an account on their website in order to get anything from them. They will charge only nominal handling fees and you will get the requested construct. There is an intermediate step involved called “MTA (Material Transfer Agreement)”. Once the formalities of MTA are over, they will dispatch the requested construct immediately (they do it internationally as well). I used their services and found them very efficient and reliable (within 3-4 working day internationally).
Web Address: http://www.addgene.org/pgvec1
3. Codon optimization:
Well, now you got a plasmid containing your gene of interest. It can be a bacterial or human gene and now you want to express it in different systems (e.g. bacterial, yeast, insect or human cell lines). For optimum expression, you need to optimize codons. Most of us are aware of bacterial codon optimization, but we can also optimize codons for any system including expression in human cell lines. I find this table quite useful especially to choose “stop codon optimization”.
Web Address: http://www.geneinfinity.org/sp/sp_codonusage.html
4. Primer designing for PCR and DNA sequencing:
You know the basics; we just need to apply it in action. I recommend using this online tool called Net-Primer.
To use this tool, you need to have two things: Free registration on their website (they are spam free as well) and latest version of Java (which is also free). The best part of this software is that it lets you “actually see the secondary structures” in the primers. Important point to remember here is that all your entered sequences (sense and antisense, forward or reverse primers) should be from 5’ to 3’ direction. Otherwise you may get false results. I refer this software to design my primers (PCR and sequencing). To access it just click “Click Here to Access NetPrimer” on the below webpage.
Web Address: http://www.premierbiosoft.com/netprimer/index.html
5. Mutagenesis (point, silent etc):
Research is all about experimenting. Isn’t it? So many times simple cloning is not sufficient. To study structure-function relationship, we may need to insert, delete or mutate amino acids. Sometimes, cloning strategy may also involve introduction of a restriction site, but not the alteration of amino acid sequence. In that case, I will recommend Wat-Cut software. This is also free online. This software tells you the possibilities of introducing point or silent mutations. Have a look, this is also easy to use.
6. DNA Tools:
I guess, using these sets of tools, you can reduce the chances of making mistakes which may amplify down the line to give you troubles in the cloning. Finally, I hope and wish that you have generated the required clone and are ready to analyze your DNA sequencing result. How exciting! At this point of time, you may need to use some group of tools (listed below):
a) Sequence cleaner:
Sometimes, you have a DNA sequence but with numbers associated with it. You can use DNA sequence cleaning programme to remove these numbers (example below).
b) Reverse/complement generator:
This programme comes in handy especially when you are doing non-directional cloning. You may get a positive clone but in the reverse orientation. Therefore, to analyze its DNA sequence result, you can use this programme. This is also useful in making reverse primer where you need to convert it from 3’ to 5’ for the use in either primer design software (above) or ordering it.
Web Address: http://www.bioinformatics.org/sms/rev_comp.html
c) Duplex generator:
If you are using pDRAW or other plasmid design software then you can easily generate DNA duplex (which comes in handy to design reverse primer). But suppose you have selected a small piece of DNA and don’t want to use pDRAW just to make that bit of DNA into duplex, then this software is for you. Just select the correct orientation, and it will give you DNA duplex.
Web Address: http://dbr.csoft.net/chem/dna.php
d) Translation tool:
I would recommend using this tool to translate your DNA. This is very useful as it offers you an option to explicitly show results with spelling “Met” and “Stop” between your single amino-acid codes.
Web Address: http://web.expasy.org/translate/
Other related tool which is useful in other sense that it gives you both DNA and protein sequence on the same page with the graphical representation of the result.
Web Address: http://insilico.ehu.es/translate/
e) Multiple sequence alignment:
I found this site useful to align multiple DNA or protein sequences. If you use multiple DNA sequences to align, then just change DNA weight matrix to ClustalW in the “step 2”. Rest of the parameters can be default for most of your routine analysis.
Web Address: http://www.ebi.ac.uk/Tools/msa/clustalw2/
The list of the above resources is not exhaustive. But this is a complete list of basic tools required for one successful cloning and a must have for molecular biologists. It is rightly said once by Winston Churchill that:
I wish you all a successful cloning (and offcourse fast)!!! Leave your thoughts below using comment form.
By the way, this is not the end of the story. Want to know all about PCRing? Watch out for my next post.