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The problems to identify mouse genotype with PCR


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#1 pine99

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Posted 22 February 2010 - 09:21 AM

I am trying to identify transgenic mouse genotype with PCR, the target gene is Hygromycin B, primers are CAA GAC CTG CCT GAA ACC GAA C and TTG TTG GAG CCG AAA TCC GC. I used Sigma Extract-N-Amp Tissue PCR kit to extract mouse tails DNA and run PCR. There are problems as followe:
1. the product band is very weak.
2. the result of the same sample is always not repeatable ( I donít think there is contamination)
3. there is primers dimmer
4. there is product smear when run the gel, it is Ok to run PCR with the same amount sample to indentify other gene.

How to solve those problems, any suggestions will be appreciated.

#2 ivanbio

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Posted 22 February 2010 - 09:33 AM

Here are some observations:

1. If you are using tail snips, make sure they are fresh. If you cut them days before and do not cut them again right before extracting the DNA, then there is a good chance you are not getting enough DNA extracted.

2. The Extract-N-Amp tissue kit is very good for tails but not as good for other fresh tissues. If you are using tissues like embryos or yolk sacs, make sure not too use too much tissue or the PCR will be inhibited (too much protein in your DNA samples).

3. Weak PCR bands can be due to a number of reasons, including PCR conditions that are sub-optimal. If you did not optimize your PCR assay, it may be a good idea to do so (using a PCR machine with gradient capabilities is great for this). Assays that have not been optimized have a tendency not to be robust, which may be the reason why your assay is not repeatable.

4. Primer dimers can form for a number of reasons, including if you store the primers together in solution, or not frozen (at 4oC instead of -20oC). Also, if you use too much primer (>1 uM final concentration) it can lead to primer dimers.

5. As for gel smears, if your DNA is degraded then you will get a smear. If your PCR conditions are too non-specific (the annealing temperature is very low, much lower than 56oC) then you may be getting non-specific amplification.

6. Finally, your PCR assay may be badly designed. Unless you know this assay works (from a colleague or a publication), all your problems may be coming from the assay itself. If that is the case, then re-designing the assay would be the solution.

Good luck!

Ivan
Carlsbad, CA




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