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

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

thanks I will try that.

appreciated

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.

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

thanks for your reply

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

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

hi all;

I found a definition for genus Prevotella saying that it was under the Bacteroides species but after 1990 it became a separate genus from the bacteroides. that's understandable!!!!!!!!!!!!!!

Using Bacteroides as a marker to identify the nonpoint and point sources of fecal contamination in the water, in these literatures they always write Bacteroides-Prevotella marker. what do they mean by that? do they mean that both the Bacteroides and prevotella species are used as markers or they still treat Prevotella as one of the Bacteroides species?

I would mostly believe the first assumption!!!!!!!!!!

your clarification is highly appreciated

thanks

You screwed yourself. Your calculation is 25 ul (PCR solution) x 400 ng/ul BSA = 10,000 ng BSA. To get 10,000 ng you need 25 ul of the BSA solution. But if you add 25 ul of BSA solution, you now have 50 ul of PCR mixture that is only 200 ng/ul of BSA. Right?

 

You need to do one of two things:

 

1. Add all your PCR reagents (in solid form) to the BSA solution you have already prepared. That would mean that the solution was 400 ng/ul of BSA.

 

2. Make up a 100x BSA solution. That would be 40 mg/ml BSA solution as your "stock". You would then add 0.25 ul of the stock BSA to your PCR solution to get 400 ng/ul.

thanks alot for the help:doh:

so do you mean that the prokaryotes consists of :

1-bacteria

2-Archea

by the way what do you mean by 'true bacteria'

thanks

ok, but i have the BSA already prepared in solution with a concentration of 400ng/ul. I prepare 25ul of PCR mixture and I did the calculations on it. the result was that 0.025ml*0.4mg/ml=0.01mg of BSA

ok, how many ul of BSA that contain 0.01mg?

I am confused

hi all

After i mix the PCR reaction mixture (DNA, primers, Master Mix) i need to add BSA (400ng/ul) how do I calculate how much i need to add of BSA to the mixture.

thanks

hi all:

In one article i found that they were successful in amplifying 16S out of eubacteria. what is the difference between the bacteria and the eubacteria and prokaryotes?

also what is the difference between the universal primer that would amplify

16S gene and the one that is specific to 16S gene? please some one define the universal primer for me?

thanks

I use 13.4ul of the DNA but it is 10^-5 dillution. the other thing is that this procedure worked for the bacterial DNA extracted from the other animals but not for the deer!!!

hi all

I need your help in this matter....I am stuck I guess!!!!

I collected manure samples from the following animals:cow, deer, pig, chicken, chicken litter. Then I amplified the 16S rDNA from each animal DNA using universal primers 515F/1492R. I could amplify the 16S rDNA gene from all of the animal samples except the deer. The PCR protocol I am using is:

94C for 10min

35 cycles of the following 3 steps:

94C for 1min

50C for 1min

72C for 1.5min

72C for 10min

4C for hold

the previous protocol worked for all animal samples except the deer sample!!!

I used Fast Prep kit to extract the DNA.

The melting temperature for 515F primer is 63.8 and for 1492R is 49.4 and as mentioned above I am using an annealing temp. of 50C . I tried to raise the temp. to 63C but I still don't see any bands. I usually load 13.4ul of the DNA sample to the master mix (total of 25ul).

If you can help me figure this out please reply .

May I please ask you if the pair of primer (Forward and Reverse) are work on one templete on the same time. meaning that one goes from 5' to 3' and the other goes from 3' to 5'?

thanks for your answer

thanks for the reply but the article you sent speaks about the Universal primer that would amplify 900bp of the 16S rDNA gene. the variable regions I am talking about are scattered along the gene and interrupted by a conserved sequences (used as primers).

Thanks for the help

I will be amplifying the variable regions for 16S rDNA and looking for Universal primers for bacteria only can some one help me with this please!!!!!!!!!!!!\

I looked up some articles but i got confused wether those primers are used for conserved regions that are the same among all bacteria or not.

thanks

hi all:

I am working with universal primers for bacteria and I came a cross some primers that are like follwing:

F984GC (position:968 to 984)

R1378 (position: 1378 to 1401)

this pair of primers is used to amplify 16S rDNA gene.

the question is that if the forward primer starts at nucleotide 968 and stops at nucleotide 984 and the reverse primer starts and finishes at different site

so, these two segments do not hybridize with each other. since the sense strand segment is different than the antisense strand segment then the amplification using this pair of primer will result in two segments.

is what I am saying correct, please tell me if I am wrong or if you can clarify more I would appreciate it.

Thanks

Thanks for your reply . The mistake I did is I thought that this gene has the same length among all bacteria!!!! if this was true then LH PCR will not be applicable.

The gel I used to separate the 16S rDNA gene (from different isolates) was based on the molecular weight so I got all the segments on the same line and when i sent the product for sequencing they returned the same segment length (~1500bp in length). If this gene is naturally different in length between bacteria then it should yield different positions on the gel (I used agarose gel). I remebered something, is the length of this gene differs among all bacteria or only eubcteria?

Thanks

your response is highly appreciated

Dear All:

I really need your help with this, please reply as soon as you read the massage !!!!!!!!!!!!!!!

I read about the L-H PCR (Length Heterogenecity PCR) which is an amplification of 16S rDNA gene and separates it based on the length of the gene. The question is how the 16S rDNA gene is having different length and how this works with the primers. I amplified 16S rDNA gene using 27f and 1492R primers and when I ran them on a gel they all lined up in one raw having the same length. I am totally confused!!!!

please help me out with this if you know any thing about it.