Single Strand Conformation polymorphism (SSCP)

[Alkendi]

hi all;

I am currently working on SSCP, capillary electrophoresis to separate DNA sample from multiple bacteria (extracted from animal feces). The profile does not show all the peaks expected but just few up to 7 peaks. I am expecting unlimited number of peaks to show up. the procedure I used is this:

-1:18 dilution with Tris Buffer

-denature for 5 minutes at 94C

-cool on ice for 5 minutes

-add the dye and leave for 5 minutes

 

the electrophoresis condition:

-inject the polymer

-wait for 10 minutes

-inject a low current (1KV) for 5 minutes

-inject the sample (at 10KV, 60s)

-separate the sample (at 12KV, 150 min)

 

 

please if you can help email me back because I have been working on this for 3 weeks and I could not get the desired results. ( I need more peaks to show up).

I appreciate your help

[CharonY]

To clarify are you trying to do SSCP with whole chromosomal DNA?

[Alkendi]

thanks for your reply

no, I am amplifying V3 region on 16S gene which is ~200bp long.

[DrDNA]

thanks for your reply

no, I am amplifying V3 region on 16S gene which is ~200bp long.

 

Besides the sample itself, your resolution is mainly based on injection load and volume, power, time, column length and polymer density.

 

1. What kind of sample prep and pre-prep are you doing? In other words, how are you removing the poop?

2. For what size DNA was the CE protocol that you are using optimized? Matrix (polymer) material and conc, time, power, etc....are all important and can be optimized.

3. What is your injection volume and have you tried less volume? Smaller volumes usually mean better resolution.

4. Have you tried injecting a less concentrated sample? Could possibly be overloading....?

**5. Do you get expected results from your standard samples? What are they and are they similar (in size and range) to what you are trying to resolve?**

6. Are all reagents fresh?

[Alkendi]

Besides the sample itself, your resolution is mainly based on injection load and volume, power, time, column length and polymer density.

 

1. What kind of sample prep and pre-prep are you doing? In other words, how are you removing the poop?

after i collect the poop I do a DNA extraction using FastPrep Kit. Then I use a pair of primers to amplify the V3 region (I use 10^5 dilution of the original sample).

2. For what size DNA was the CE protocol that you are using optimized? Matrix (polymer) material and conc, time, power, etc....are all important and can be optimized.

the CE should work between 200bp and 300bp lengty. we use 7% pure polymer. I do polymer injection for 40min at 80psi, inject the sample for 60s at 10kv, separation at 12kv for 150min.

3. What is your injection volume and have you tried less volume? Smaller volumes usually mean better resolution.

I used 150ul, 200ul and 300ul

4. Have you tried injecting a less concentrated sample? Could possibly be overloading....?

yes, I tried 1:18, 1:100

**5. Do you get expected results from your standard samples? What are they and are they similar (in size and range) to what you are trying to resolve?**

we do use the 50bp ladder as a standard and when using 1:1polymer many peaks showed up in the profile but then current started to drop at some point. we also used 100bp ladder and few peaks showed only at pure polymer and many showed when 1:1 polymer used.

6. Are all reagents fresh?

I just began to work on CE (in a month) so all reagents are recent.

Our problem is that even when we use a standard sample like the DNA ladder we get only few peaks and then a flat line.

 

I wish you can help after these clarification.....I am stuck and i'm really desperate for help

 

I appreciate your concern

[DrDNA]

I just began to work on CE (in a month) so all reagents are recent.

Our problem is that even when we use a standard sample like the DNA ladder we get only few peaks and then a flat line.

 

I wish you can help after these clarification.....I am stuck and i'm really desperate for help

 

I appreciate your concern

 

Ah ha, your standards not working correctly is a REALLY important bit of info.....it apparently has nothing to do with your particular unique sample.....you've got to get it worked out with the ladder first obviously or you can never hope to resolve your sample.

 

OK, let's go down the list...

Did it ever resolve the ladder correctly? If so, when? By whom?

What protocl did they use: ....injection volumes, concentration, size and range of size of the ladders they (or you) used when it worked.......etc??

Have you tried other reagent stocks, (buffers, polymer, etc...)? Can you get your hands on some other stocks?

 

EDIT: I just saw your answers mixed in with my quote.

Re: ":using 1:1polymer many peaks showed up in the profile but then current started to drop at some point. we also used 100bp ladder and few peaks showed only at pure polymer and many showed when 1:1 polymer used."

Which are you using now? 1:1 or pure?

I wouldn't worry too much about current dropping if you are getting resolution.

[Alkendi]

OK, let's go down the list...

Did it ever resolve the ladder correctly? If so, when? By whom?

the most successful run I've ever seen since we began to use the EC is when we used the 100bp ladder with 1:1 polymer (but as i said the current droped at some point). the other student who run this was hesitant about the current dropping but I said that since we see nice peaks we should not worry about the current that much!!!I'm I right?

What protocl did they use: ....injection volumes, concentration, size and range of size of the ladders they (or you) used when it worked.......etc??

the student did not do any dillutions on the 100bp ladder stock but I know that she loads between 150-300ul in the EC. I don't know about the concentration but I can get it for you. the size of the ladder suppose to be increamental starting from 100bp,200bp, 400bp,...etc.I think it is up to 1000bp or more.

Have you tried other reagent stocks, (buffers, polymer, etc...)? Can you get your hands on some other stocks?

this is the only polymer that we are using (7% polymer) and for separation we use TE buffer mixed with 10% glycerol.

Which are you using now? 1:1 or pure?

I am using the pure polymer.

I wouldn't worry too much about current dropping if you are getting resolution.

[DrDNA]

You must replicate the EXACT conditions that were used when it worked correctly. You need to go back to square one ....start with new bottles of polymer, buffers, glycerol, etc.

 

 

The CE system is not complex. It is not mysterious. Not much besides cleanliness, the injector, the sample, reagents (matrix and buffer) and power can go wrong.

 

It is just a thin tube and high voltage electrical system with an injector at one end. Everything else is important but disposable and can be easily changed.

 

Is the system clean? What about the injector (check manual for cleaning or servicing)?

 

Do you have a manual? Does it have a troubleshooting section?

What does the manual say about current dropping and pure vs 1:1 or other ratios? In my experience, the current changes all the time depending on what you load it with and inject (of course ionic strength effects resisitivity, conductivity, etc an therefore current).

[Alkendi]

thanks I will try that.

appreciated

[DrDNA]

Let me know how it goes.

[Alkendi]

hi DrDNA

I am currently working on the 100bp DNA ladder (dsDNA) and it should produce 12 peaks on the CE. yesterday the profile showed 12 peaks!!!but all students in the lab are facing the problem of reproducibility, the result is not reproducible. After i got the sucssessful result I ran the same sample once again under similar conditions and I only generated one peak!! and I tried again after i changed the buffer and still one peak shows up.

By the way I am running the ladder in buffer only without polymer.

what could be wrong, I cannot get reproducible result for the same sample under similar condition??!!! which is very strange.

if that would help you think with me I use a flourescen dye (OliGreen) to tag the DNA and it is used for both dsDNA and ssDNA. In this case i am using it to tag a dsDNA. the process is I add enough amount of the dye to the DNA (1:1 ratio) and leave it for 5 minutes then run it on CE. During the 5 minute wait I wrap the tube with aluminum foile as the dye is light sensitive.

 

please think with me, what could be that I did not fix???

 

appreciate your help