Basic of the basic query! - (Oct/11/2008 )

hi all
I have to do certain things independently (finally!!!) but with nil guidance....will be doing for the first time also

Would like to ask very basic questions. if anyone can help.....please

1. Need to prepare a vector (for 3 piece ligation??)
a) what all steps should be followed from the starting-say I received an aliquot of 2 microliter of plasmid. Now how to go forward with it.

Just for making things clear, I have to cut with 2 restriction enzymes (BamHI & NotI)

2. Amplify 2 PCR products of 1 kb each.
a) one product is with BamHI AND Kpn site
another with Kpn and NotI site

3. Finally then 3-Piece Ligation

i very new to molecular techniques
any help will be appreciated

ps: i'm basically concerned with how much concentrated should be the plasmid and vector till it reach the final stage...or in another words say how much in ul???

It important to have enough starting vector to work with and to go back too in the event of ligation failure (where more vector need to be cut). So the first thing that you need to do is make more of your vector. Transform the vector you have into a cloning e coli strain.

As E.coli plasmid transformation is very efficient, make a 1:100 dilution with the plasmid stock you have and transform about 1ul of the dilution. Conduct the transformation (electroporation or chemical transformation). Dilute the transformation mix with SOC and recover for 30minutes. Plate 10ul, 50ul and the result of the cell mix onto selection plates.

Pick 2 of the resulting colonies. Grow them in two 100ml cultures. Check that the plasmid is structurally correct and you do have the plasmid you are suppose to have. People can make mistakes. That vector in your hands may not be the right thing.

(Yes, I am wasteful. 100ml culture is larger than you need. And you could in theory build the plasmid with the 2ul of vector given. But I believe it is better to do a good job once, and never return. Also it is better to be safe than sorry.)

Once you have confirm that the plasmid is correct, proceed to cut the vector with the appropriate enzymes. I would cut 15 - 20ug of vector. This is extremely wasteful but I think it is better to cut once, and have enough DNA to waste and if needed too make multiple ligation attempts.

Next concerning your PCR fragment, look up NEB technical guide. Restriction enzyme require a few bp surrounding both ends of the restriction site before the restriction enzyme can cut the site efficiently. The rule of the thumb is 6bp but some enzymes need more and others need less. NotI requires a minimum of 8bp while BamHI can with 2bp (But I would give it 4bp).

A useful tool to design primer is the Nortwestern oligocalculator. It find out the melting temperature ™ of the primer and checks for the presence of hairpin loop structures and self complementation. Only the section of the primer that actually binds to the template is considered for the tm calculation.

For PCR fragments below 5kb, I would use KOD hifi. It is a relatively cheap proof reading polymerase and has high yields for PCR products.

Lastly for a 3 way ligation, the molar ratio between fragments is 1:1:1. Use a nanodrop (if you can find one) to calculate the concentration of the DNA fragments. Ligate at 16 celsius. Do not use quick ligase (which contain PEG). You can use colony PCR to help screen the colonies. If you do use colony PCR, amplify across junctions between DNA fragments. Onlu these junctions are unique to a properly constructed plasmid. PCR is sensitive enough to amplify left over DNA from the ligation mix on the gel. So amplification of a DNA fragment (and not the junctions) can amplify this leftover DNA leading to false positive signals.

Nanodrop is a spectrophotometer that can measure the DNA concentration of a small sample of DNA (1ul).

Colony PCR [attachment=5422:Bacteria...lony_PCR.doc]; Here is my protocol of Colony PCR. There are variations of this protocol, which you can find by doing a search in this Forum or a search via google.