Why is 3 mg Too Much for PCR Reaction?

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In summary, the conversation discusses the use of 3mg instead of 10ng template for PCR reaction and the lack of product when the gel was run. The potential reasons for this include too much DNA being inhibitory due to competition for annealing of primers and dNTPs, increased probability of nonspecific primer binding, and potential contamination from the DNA isolation method. It is recommended to use a lower amount of DNA, preferably between 10ng-500ng, for more efficient and accurate PCR results.
  • #1
karthik3k
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I used 3mg intsead of 10ng template for PCR reaction.

And i didnt find any product when i ran it on a gel.

Can anybody explain why this happens ?

Plz don't say its due to impurities...
Iam quiet sure abt the sample i used ...

THanx
 
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  • #2
You mean 3 ng? You are on the low end, but it still should be enough. First questions: did the PCR work before, did the sample work before, did you include positive controls in the protocol?
 
  • #3
Yes.
I used 3 micro gram
 
  • #4
Too much DNA template is inhibitory due to competition for annealing of primers and dNTPs. So you end up with a lot of short fragment because dNTPs is depleted quickly. It should also be noted that the probability of nonspecific primer binding is increased due to change of the optimal quality to a suboptimal quality.

Also large volume of template migth carry "garbage" from the DNA isolation step, depending on the methode use and you. This "garbage" migth inhibit the enzyme.
 
  • #5
karthik3k said:
I used 3 micro gram
Ah, you can tell from my asumption it must've been 3 ng.. that 3 mg really is way too much :smile: the normal range would be 10 ng - 500 ng, less is better since you get less contaminants and the reaction overall is more efficient.

Ian said it well, your primers are binding all over the genome since there is so much DNA to bind to, your dNTPs get depleted, and you are introducing contaminants by adding such a large volume (remember DNA binds proteins).
 

1. Why did no product form during PCR?

There could be several reasons for this. It could be due to poor quality or degraded DNA template, incorrect primer design, inadequate enzyme activity, or contamination in the reaction components.

2. How can I troubleshoot if no product is forming in PCR?

First, check the quality and integrity of the DNA template. Then, verify the primer sequences and ensure they are specific to the target gene. You can also try adjusting the PCR conditions, such as the annealing temperature or cycle number. Finally, check for contamination in the reaction components.

3. Is it possible to have no product formed in PCR even with good quality DNA and proper primer design?

Yes, it is possible. Other factors such as incorrect enzyme concentration, presence of inhibitors, or low enzyme activity can also affect PCR amplification.

4. What can I do if I repeatedly get no product in PCR?

If you consistently get no product in PCR, it may be helpful to troubleshoot by changing one variable at a time. This could include using a different DNA template, adjusting the primer concentration, or trying a different PCR enzyme.

5. Can using too much or too little DNA template affect PCR product formation?

Yes, using too much or too little DNA template can affect PCR product formation. Too much DNA can lead to non-specific amplification, while too little DNA may not provide enough template for successful amplification.

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