ligation transformation of puc18 and phage lambda using ecor1 and bamh1 - general double/single digest problem (Dec/06/2005 )

I single digested phage lambda DNA in my lab and then attempted to insert it into a puc18 plasmid. So after that I plated and received positive colonies.
I then lysate the bacterium and extract the vectors once the bacteria have proliferated and created more and more of the plasmid. When I then take the transformation/ligation product I treat it with ecor1 and then double digest with bamh1 & ecor1. this takes place in two separate evaluations, a tube with single digest & a tube with double digest. When i gel these two samples I get no signatures at all for the single digest ?
For the double digest I get very weak signatures on the gel ?

Exactly what would i have done wrong, or what where is my error coming into play ?

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Well I guess you would have to explain yourself a bit better cause here I cannot really get what you're doing

You want to make single and double digest of
1/pUC alone ?
2/ Lambda DNA alone ?
3/ Some generated clones from a transformation/ligation of LAmbda DNA cloned into a pUC vector

More explanations are needed to help you !

Pesji

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choice 3.

I did a single digest of puc18 ligated with a ecoR1 treated phage lambda fragment.
I then performed a double digest with the same vector ligated with the phage lambda DNA fragment.
So when i separately gelled both the single and double digest members(positive control, negative control, positive colonies) the single digest had no residue show up on it. the double digest showed some residues in the positive control but it was very streaky and extremely faint you have to strain your eyes to notice it. Actually the positive control showed a lighter streak(molecular weight) then the actual positive colonies which showed higher molcular weight streaks ? How is it possible of this happening I followed strict instructions of a kit so I wasn't just randomly screwing up enzyme/vector ratio. was the error in the lysation of the bacterial membrane ? actual gel ? not an adequate number of regenerated plasmid in the e coli ? Any help would be extremely useful for me to reatrce my steps.

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Well it's getting a bit better but my understanding of your post is still prettty low Sorry

let's try to help
You did a transformation of pUC vector that you ligate with a big insert obtained from Lamda phage DNA
Up to now it's clear !

You extract the DNA out of the recombinant colonies after culturing them in liquid I suppose

Still right ?

Then you run a restriction digest of your clones DNA: single or double

1/ First question your vector was originally cut with EcoRI yes ? You say you treat the Lambda DNA with Eco RI you mean that you digest it with EcoRI and recover the fragment ?

2/ How big is the EcoRI lambda insert ?

3/ Why do you want to use BamHI ? Is it cutting inside your insert ?

The control should show only the vector (pUC ) band

Positive control ? What did you use ? Lambda DNA ? Plasmid ?

Pesji

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