3 replies to this topic

[george@CASE]

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  

[predoc]

I do a Qiagen Midi-prep and it works out to give me a conc of atleast 1ug/ul. I did a 50ml prep.

Hope this helps. BTW, I have question for you: do you know if there is an ATg upstream of the MCS in this vector for in-frame cloning. I checked on MAcvector and it shows me stop codons in all frmaes upstream of the MCS. Does this mean that you can clone anyhow?

Thanks!!

george@CASE, on Mar 30 2005, 02:26 PM, said:

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  

[predoc]

God! Dod I reply to a 2005 post?

Goodness, I must be going nuts!

predoc, on Feb 18 2009, 02:01 PM, said:

I do a Qiagen Midi-prep and it works out to give me a conc of atleast 1ug/ul. I did a 50ml prep.

Hope this helps. BTW, I have question for you: do you know if there is an ATg upstream of the MCS in this vector for in-frame cloning. I checked on MAcvector and it shows me stop codons in all frmaes upstream of the MCS. Does this mean that you can clone anyhow?

Thanks!!

george@CASE, on Mar 30 2005, 02:26 PM, said:

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  

[perneseblue]

predoc, on Feb 18 2009, 01:02 PM, said:

God! Dod I reply to a 2005 post?

Goodness, I must be going nuts!