User:Anthony Salvagno/Notebook/Research/2010/03/09/Project Lambda Research
I have classes today so productivity is going to drop. I'll try and keep up the good work though. Yesterday I gathered intel on what I need to do and what supplies I'm going to need. Today I intend to do whatever comes my way, but I first need to understand a few things. I want to understand the basics of the Klenow fill-in reaction. I also want to check out a bunch of protocols and decide the best course of action to take.
Klenow Fill-in Reaction
Most websites I've looked at say the same thing. With every manual, it says Klenow fragment is used for 5'->3' polymerization and will leave a single base overhang. If you don't have exo- version, then the single base overhang can be removed, but otherwise you are stuck.
From Protocol Online:
- Fill-in Reaction - I added 2 uL of Klenow (NEB: 3'-5' exo-) and 1 uL of 2.5 mM dNTPs and incubated at 37 C for 30 minutes. I then heat inactivated Klenow by heating at 75 C for 20 minutes.
According to NEB:
- Reaction Conditions: 1X NEBuffer 2, Supplemented with 33 μM dNTPs (not included), Incubate at 37°C.
They also say that 1 unit "is defined as the amount of enzyme required to convert 10 nmol of dNTPs to an acid-insoluble material in 30 minutes at 37°C." Looks like I'll have to do a little math. This is hard for my rapidly decaying physics brain.
Blunt end Reaction here. Looks really simple as Klenow buffer, dNTPs, and DNA are all that's needed. Just let reaction go for about 30 min and voila! They also prescribe heat inactivation. Doesn't say anything about purification, but I'm sure normal reactions would work well with Qiagen kit and lambda would do alright with ethanol precipitation.
As for the heat inactivation, I've seen other protocols that say you can use EDTA to stop the reaction. This might be a better approach.
Also see here. They point me to the source that I was going to go to next, Molecular Cloning. They say (from MC) that phenol/chloroform extraction is good deactivation.
Also see Molecular Cloning pg 9.54:
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