14 replies to this topic

[brightfield]

We bought two plasmids from Clontech to be used for controls in our imaging experiments. One is AcGFP and the other is DsRed, both targeted to cell membranes, and driven by CMV promoter. We use electroporation to transfect avian embryos. The AcGFP seems to have complete lack of expression - confirmed by flow cytometry on cells transiently transfected using the Amaxa system. The DsRed has a very small amount of expression, but compared to other plasmids we have used in the past with CMV promoter, I would estimate the efficiency lower by at least ten-fold. Certainly, neither of these can be used for imaging.

We've been troubleshooting for weeks, and running out of ideas. We know it's not a matter of our transfection system, because we can electroporate a different plasmid (H2B-GFP) very effectively. I thought my student may be overloading the Qiagen columns in the MaxiPrep, but she ran it using lower volumes this week, and the results did not change. According to a NanoDrop spec, the amount of DNA that was purified was quite high. Tomorrow, my student is going to run a gel on the samples Qiagen recommends to take throughout the MaxiPrep protocol for troubleshooting.

Does anyone have advice/suggestions? My basic assumption is that these plasmids are routinely used in widely different systems, and would be great for co-transfection experiments. Am I missing something?

[AquaPlasmid]

1) Did you check endotoxin contamination of the prep?
2) Electroporation kills a lot of cells and might worth trying lipid based transfection as well.
3) Always check your plasmid in the gel, spec often overestimates.

[brightfield]

AquaPlasmid, on Jan 29 2009, 09:18 PM, said:

1) Did you check endotoxin contamination of the prep?
2) Electroporation kills a lot of cells and might worth trying lipid based transfection as well.
3) Always check your plasmid in the gel, spec often overestimates.

1. We are using an endo-free kit, but I am concerned about endotoxins. However, I have used this kit in the past many times and never ran into problems like this. Do you have advice about how to check for endotoxins? Also, would this result in an otherwise good plasmid completely not working?

2. I realize electroporation can harm cells, but our embryos develop normally. I've actually published a paper within the last few months using this technique. Unfortunately, lipid transfection is much less efficient than electroporation. So, as I said in my post, I don't think transfection is the issue. We are able to successfully electroporate other plasmids.

3. Yes, we are doing this tomorrow, as I said.

Next week, we're going to try some other constructs that I ordered from AddGene. Hopefully, we'll have better luck.

Edited by brightfield, 29 January 2009 - 05:49 PM.

[perneseblue]

hmmm have you checked the basics?

Is the plasmid correctly constructed. Has it been checked by restriction site mapping, and sequencing. Has there been any mutation in the promoter, kozak sequence, or gene.

[brightfield]

perneseblue, on Jan 30 2009, 12:15 AM, said:

hmmm have you checked the basics?

Is the plasmid correctly constructed. Has it been checked by restriction site mapping, and sequencing. Has there been any mutation in the promoter, kozak sequence, or gene.

Given that these two plasmids were ordered directly from Clontech and prepped within a day or so of arrival, do you really think this is a possibility? On two different plasmids? I'm not trying to be antagonistic, as I've had the same thought. It's just that I've asked around, and been told that's extremely unlikely. Is there something that can easily induce mutations that I should be aware of?

We are planning to do the restriction cut. Maybe we should do the sequencing just to be sure.

Edited by brightfield, 30 January 2009 - 05:30 AM.

[perneseblue]

Very unlikely, but you never know and the plasmids are giving trouble.
It maybe a 1 in a million event but you could be that unlucky one. No harm checking.
Sanger has once given me a bad cosmid before.

[AquaPlasmid]

brightfield, on Jan 29 2009, 07:48 PM, said:

1. We are using an endo-free kit, but I am concerned about endotoxins. However, I have used this kit in the past many times and never ran into problems like this. Do you have advice about how to check for endotoxins? Also, would this result in an otherwise good plasmid completely not working?

We use LAL for endotoxin detection. Since you're using endo-free kit, that might not be the problem. Is H2B-GFP also driven by CMV promoter? CMV sometimes works strong in some lines but not others.

[labrat612]

Is this the first time that you are using the Amaxa system with this line?? If so, perhaps using a different method with this line would be helpful. Or use a positive control with the system and your line to determine the precise source.

[brightfield]

AquaPlasmid, on Jan 30 2009, 12:57 PM, said:

brightfield, on Jan 29 2009, 07:48 PM, said:

1. We are using an endo-free kit, but I am concerned about endotoxins. However, I have used this kit in the past many times and never ran into problems like this. Do you have advice about how to check for endotoxins? Also, would this result in an otherwise good plasmid completely not working?

We use LAL for endotoxin detection. Since you're using endo-free kit, that might not be the problem. Is H2B-GFP also driven by CMV promoter? CMV sometimes works strong in some lines but not others.

Good question. I think the H2B-GFP is actually made from a pCAG vector, similar (or exactly) like this one on AddGene:

http://www.addgene.o...n...&cmd=findpl

However, in the past, we have also used plasmids that used the old pEGFP vectors from Clontech which have CMV promoter and were working in our system.

We have two pCAG vectors that we are going to try next week. Hopefully, we will have better luck with at least one of them.

[scolix]

We also had problems with Dsred from clontech. It was quite weak. The CMV with AcGFP worked but not as efficiently as I would have preferred. It was weak. I had cloned them into a different vector before I could use the acGFP. Dsred was trashed, not literally.

[brightfield]

scolix, on Feb 1 2009, 12:04 PM, said:

We also had problems with Dsred from clontech. It was quite weak. The CMV with AcGFP worked but not as efficiently as I would have preferred. It was weak. I had cloned them into a different vector before I could use the acGFP. Dsred was trashed, not literally.

Interesting. I ordered a pCAG-DsRed from AddGene. Hopefully, the CAG promoter will give much higher efficiency. What vector did you end up using?

Edited by brightfield, 01 February 2009 - 01:33 PM.

[brightfield]

I had my student run a gel on one of the Clontech plasmids (CMV-AcGFP) that is not working and an older plasmid (CMV-Rac1-GFP) that was working several months ago, but now is not working. It doesn't look like a great gel, but I think you can see that the linearized DNA migrates to approximately the correct size (5 kb). However, the uncut DNA appears to be primarily in two bands, which I assume the supercoiled form migrates lower (faster). Unfortunately, it is the other band in both instances that appears brighter. Does this indicate there is indeed a problem with our prep?

[brightfield]

Can anyone help me with this? It seems the uncut DNA is running slower than the linearized DNA - that's not good, right? What kinds of things could cause this outcome.

[AquaPlasmid]

brightfield, on Feb 7 2009, 01:40 PM, said:

Can anyone help me with this? It seems the uncut DNA is running slower than the linearized DNA - that's not good, right? What kinds of things could cause this outcome.

Often linearized plasmid runs between supercoiled and relaxed, but for large plasmids, linearised can run faster. It may varied whether the plasmid is associated with or without salts, mono and divalent ions. I don't think you can tell if there is a problem with your plasmids with a single cut. You need to sequence it, but I double it's the expression problem you are having.

To your question on other post -"if prechilling the cell culture is needed before centrifuge," the answer is "No."

[brightfield]

AquaPlasmid, on Feb 7 2009, 04:58 PM, said:

brightfield, on Feb 7 2009, 01:40 PM, said:

Can anyone help me with this? It seems the uncut DNA is running slower than the linearized DNA - that's not good, right? What kinds of things could cause this outcome.

Often linearized plasmid runs between supercoiled and relaxed, but for large plasmids, linearised can run faster. It may varied whether the plasmid is associated with or without salts, mono and divalent ions. I don't think you can tell if there is a problem with your plasmids with a single cut. You need to sequence it, but I double it's the expression problem you are having.

To your question on other post -"if prechilling the cell culture is needed before centrifuge," the answer is "No."

These two plasmids are only ~5 kb.