Insert/vector ratio for ligation - (Aug/19/2008 )
this is my first time cloning shRNA into a vector. Normally for a ligation I would use a 2:1 or 3:1 ratio using the formula ng of vector x size of insert / size if vector and all that x ratio insert/vector. The thing is my vector is 6 kbp and my shRNA is 60bp. According to this formula Id have to use a couple of nanograms of insert for 50ng of vector. Is this ok?
Someone in my lab, my superior sorta told me to use 50ng of vector and 2 micrograms of insert, its the complete opposite of what Ive done before and it seems like a gigantic overdose of insert??
Comments please? This is my second week in the PhD and this lab is a bit confusing!
generally when ligating inserts and vectors of normal sizes, (>250bp) you can use the normal molar ratios. Often times people will quote a molar insert to vector ratio of 3:1.
However as if you go about experimenting, you will find instead that the concentration of DNA within the ligation mix is actually more important to a successful ligation, aka how likely if your vector going to find insert. And a 1:1 insert to vector ratio works (Which is more convenient and in my opinion more efficient).
Ligation is "easy" until you start ligations product exceed 10kb. At around this size, you will find that the ligation become a little more difficult.
However when you ligate very short oligonucleotide (<100bp), you will find the ligation also becomes rather difficult if you use "normal" insert to vector ratio. As your labmate has correctly pointed out, the only way to get these short inserts into the vector is to flood the ligation mix with inserts. So yes, you need a giant mega dose of insert, else the ligation will not work. If you add as much short insert as possible into the ligation mix, the reaction become efficient.
Also note that you can use colony PCR to screen your ecoli colonies. (Always amplify across the junction between insert-vector) Or in your case, use primers that bind to vector, near the insert and PCR across the insert. Run the PCR products on a 3% agarose gel and you should see a difference between vector, and vector+insert
If you insert is relatively large you can use crack solution to screen (ie you lyse the cell, and compare the plasmid on a gel. The vector+insert plasmid is larger than the vector plasmid alone)
Lastly you can insert multiple inserts into a vector at the sametime (multiway ligation). If the inserts are designed to carry incompetible restriction site overhangs at their 5' and 3' ends, multiway ligation is relative "easy".
And always talk and consult with your senior labmate. A lot of things in molecular biology takes practice. And a number of things can go wrong. And there is abit of black art about molecular biology.
P.S: conduct you ligations in a 0.2ml microtube. the ligation mix should have a volume of at least 20ul. Why? because it helps cut down evaporation. And when you precipitate your DNA (Assuming you use an electroporator in your transformations), the small tube makes it easier to see your DNA. On the same note, use dextran if you are worried of losing your ligation pellet.
Thank you so much! She was sick yesterday and wasnt here so I couldnt ask. Also shes not really my supervisor, I kinda dont have one except maybe the technical assistant who is really old-fashioned. As soon as I mentioned siRNA she lifted her hands up in the air and stated she had never done that before...so that was that lol
I designed a primer pair that should allow me to see a difference between vector and vector+insert. Tested them and optimised annealing temperature I will have either 190bp or 155bp which I should be able to see on a 2% gel.
I will use the overdose for the ligation and before that I will run a 3%gel to see if my primers annealed properly.........fingers crossed!
and best of luck.
after ligating your insert into the vector, make sure you always sequence the insert. (And have at least 2 isolates of the same plasmid, just in case one is wrong... I keep 3 but I may just be excessive)