4 replies to this topic

[cnatstop]

Hi everyone,

I'm having a tough time trying to insert ~4kb gene into pENTR/D TOPO vector. After TOPO reaction, I've been consistently getting many dozens of colonies on the plate. When I do colony PCR using M13 forward/reverse primers, I'm getting ~300 bp which indicates empty pENTR/D TOPO vector. I've screened upto 20 colonies of one TOPO rxn plate, and they were all ~300 bps. I'm wondering if I should be screening more colonies of this plate or start TOPO reaction altogether.

I've heard that screening 4-8 colonies should yield at least one colony... has anyone screened much more to get the vector w/ gene of interest?

Is there more efficient way of screening large numbers of colonies at once? A fellow student told me I could try swabbing 4 colonies per tube and at least narrow down which pool of colonies contain the one w/ gene.

Any advice would be appreciated!

[ram]

cnatstop, on 05 September 2010 - 12:11 PM, said:

Hi everyone,

I'm having a tough time trying to insert ~4kb gene into pENTR/D TOPO vector. After TOPO reaction, I've been consistently getting many dozens of colonies on the plate. When I do colony PCR using M13 forward/reverse primers, I'm getting ~300 bp which indicates empty pENTR/D TOPO vector. I've screened upto 20 colonies of one TOPO rxn plate, and they were all ~300 bps. I'm wondering if I should be screening more colonies of this plate or start TOPO reaction altogether.

I've heard that screening 4-8 colonies should yield at least one colony... has anyone screened much more to get the vector w/ gene of interest?

Is there more efficient way of screening large numbers of colonies at once? A fellow student told me I could try swabbing 4 colonies per tube and at least narrow down which pool of colonies contain the one w/ gene.

Any advice would be appreciated!

Once, I got one positive colony after screening 30 colonies. It served my purpose and I didn't have to do costly TOPO ligation again! So I will suggest to screen few more colonies!
I dont have any experience with combining few colonies in single PCR, but its a good idea, especially if u know that u have to screen many colonies to get a positive. If you get any positive PCR, u can go back to those 4 colonies and again do the PCR separately

[moerae]

I've just recently picked up to 50+ colonies and none of these have anything. I think at some point you need to put in a cut-off and start again. My next option was to combine several colonies in the same PCR reaction and go from there, but having to pick more than 50 colonies was getting ridiculous.

[ram]

moerae, on 20 September 2010 - 06:46 PM, said:

I've just recently picked up to 50+ colonies and none of these have anything. I think at some point you need to put in a cut-off and start again. My next option was to combine several colonies in the same PCR reaction and go from there, but having to pick more than 50 colonies was getting ridiculous.

Yes, I know its rediculous! Choice is yours, to put money for PCR reagents on taking a chance to screen more colonies or to invest something on doing a new ligation! But now I think you should go for the second? If you decide to do so, then you can use smaller amount of vector then what has been told in the manual and then what u used last time. This reduces number of colonies and increases chances of getting poitive; it seems that you had many colonies in earlier attempt!

[moerae]

ram, on 21 September 2010 - 12:18 AM, said:

moerae, on 20 September 2010 - 06:46 PM, said:

I've just recently picked up to 50+ colonies and none of these have anything. I think at some point you need to put in a cut-off and start again. My next option was to combine several colonies in the same PCR reaction and go from there, but having to pick more than 50 colonies was getting ridiculous.

Yes, I know its rediculous! Choice is yours, to put money for PCR reagents on taking a chance to screen more colonies or to invest something on doing a new ligation! But now I think you should go for the second? If you decide to do so, then you can use smaller amount of vector then what has been told in the manual and then what u used last time. This reduces number of colonies and increases chances of getting poitive; it seems that you had many colonies in earlier attempt!

Yeah I'm going to do a new ligation.... I've checked the ligation itself via PCR and my insert is in there... but I think it's at such a low number that the other self-ligated plasmids have gone into the cells instead. Frustrating as hell.