Cloning Difficulty - Problems with digestion/ligation (Jun/24/2011 )

I am currently trying to insert a ~650kb PCR amplified insert into a pGLUC-Basic vector (4.92KB). I have tried many different methods following manufacture protocols, all without success. My transformation efficiency is zero, with no colonies formed after 24 hours. I typically run a 50uL double digestion using the following:

Digest using:
1ug DNA
1uL/10U of EcoRV
0.25uL/10U of BamHI (concentrated)
0.5μL (0.1mg/mL) BSA
5uL NEBuffer 3
Fill to 50uL with dH2O
Digest at 37C for 1-1.5 hours

CIAP Treat Vector
PNK Treat Insert

Ligate using:
50ng Dephosphroylated vector
100ng Phosphorylated insert
2μL 10x Ligation Buffer
5μL T4 DNA ligase
Fill to 20uL with dH2O
Ligate at 16C overnight

Transform in DH5(alpha) cells

I just wanted to ask if anyone has some advice/experience using either of these digestion enzymes or the pGLUC-Basic vector.

That's way too much ligase in your ligation reaction. You need about 0.2 ul of ligase (or less). But that is probably not the problem. I'd vote for CIAP treatment of your vector as your likely issue. Are you sure you need to do this? Are you sure you need to do a blunt end ligation? This would not be my first choice (or my second).

Maybe you can extend the digestion time up to 2hours...

here is my simplified method for digestion and ligation, so far it always give good result,

I use all in ul(to ease u work)
digestion..it diluted one but it work well
plasmid 12ul
NE buff 1ul
BSA 0.50ul
Renzyme 0.5ul
dh20 up to 30 even sometimes it work for up to 50, but it depend on how conc your plasmid is..
incubate at 37C 2 hours
if u have diluted one, just follow the manual book provided by manufacturer..Biolabs

Ligation
digested plasmid 3ul
fragments (digestd) 9ul
lig buff if u use 10x 1ul or if 5x 2ul, 2x u need 5x
T4 ligase 1ul
dh20 up to 20ul.............put it at 16C, overnight is okay...for me no overligation terms we used but over digestion yes, it happen if u digest for long time...

try to use control and check your transformation methd too, if u use TSS for transformation, make sure it's good

Evanescence: That's a bad digestion strategy. Your restriction buffer is 3-5 times too dilute to work well. The buffer concentrations are important to get efficient digestion. I'm sure it works somewhat, because the enzymes are active across a wide range of conditions, but I don't see why you would choose to do the reaction at other than the recommended buffer concentration. Similarly, you are using 2x less ligation buffer than you should be using, if you are doing 20 ul ligation reactions.