copy number of pUAST - need info to troubleshoot maxiprep yield (Mar/30/2005 )

Hello everyone,

My turn to ask.

pUAST is a transformation vector for Drosophila. It is 9kb in size, and based on the pCASPER vector family.

My insert is 9.7kb in size ( I know it's big, and its hell to work with). So the whole thing is around 19kb.

Firstly, does anyone know the copy number of pUAST?

Secondly, with a plasmid this big, is it better to assume that the plasmid is low copy simply because of the huge size?

I did a maxiprep using Qiagen Maxifilter, and boy was I surprised that I didn't get a visible DNA pellet in the isopropanol step. I continued with the protocol, and running my final dna solution on a gel, I see that I did get DNA but nothing close to the yield from a maxiprep.

I was told that it was high copy so I grew up 100mL LB culture as per Qiagen protocol. This may be my mistake.

Any inputs?

Thanks.

P.S. I'm afraid to run another maxiprep. The kit is good for only 10 applications . My boss is gonna eat me alive if I use it up

*bump* sorry, that's the only time i'm gonna do that. I really need input on this.

Hello everyone,

My turn to ask.

pUAST is a transformation vector for Drosophila. It is 9kb in size, and based on the pCASPER vector family.

My insert is 9.7kb in size ( I know it's big, and its hell to work with). So the whole thing is around 19kb.

Firstly, does anyone know the copy number of pUAST?

Secondly, with a plasmid this big, is it better to assume that the plasmid is low copy simply because of the huge size?

I did a maxiprep using Qiagen Maxifilter, and boy was I surprised that I didn't get a visible DNA pellet in the isopropanol step. I continued with the protocol, and running my final dna solution on a gel, I see that I did get DNA but nothing close to the yield from a maxiprep.

I was told that it was high copy so I grew up 100mL LB culture as per Qiagen protocol. This may be my mistake.

Any inputs?

Thanks.

P.S. I'm afraid to run another maxiprep. The kit is good for only 10 applications  . My boss is gonna eat me alive if I use it up 

hello amila,

I used Qiagen's QiaFilter Maxi kit. I really don't see where I can lose my DNA as I followed the protocol to the letter.

Only reason I can think of is that the elution isn't efficient and I'm not getting anything out of the Qiatip.

Has anyone ever had problems like these in maxipreps?

hi
i did use columns for maxi prep. Due to the fact i worked with low copy plasmid, i increased the volume f solution and lysis buffer. But after load the mixture filtered on the column and do the protocol as said, i got...... close to zero DNA.
The supposed mistake was that columns were for 50ml of ulture and i did with 200ml. So i would have use 4 columns. But i can't afford that my boss would eat me too. That's why i know prepare my plasmids with the classical "home made" miniprep.
If i want to make a transcription from my sample, i dilute about 100µg in buffers 1, 2 and 3 and load it on the column. And then proceed as said by the manual. It worked good regarding purity.

Hello everyone,

My turn to ask.

pUAST is a transformation vector for Drosophila. It is 9kb in size, and based on the pCASPER vector family.

My insert is 9.7kb in size ( I know it's big, and its hell to work with). So the whole thing is around 19kb.

Firstly, does anyone know the copy number of pUAST?

Secondly, with a plasmid this big, is it better to assume that the plasmid is low copy simply because of the huge size?

I did a maxiprep using Qiagen Maxifilter, and boy was I surprised that I didn't get a visible DNA pellet in the isopropanol step. I continued with the protocol, and running my final dna solution on a gel, I see that I did get DNA but nothing close to the yield from a maxiprep.

I was told that it was high copy so I grew up 100mL LB culture as per Qiagen protocol. This may be my mistake.

Any inputs?

Thanks.

P.S. I'm afraid to run another maxiprep. The kit is good for only 10 applications  . My boss is gonna eat me alive if I use it up