I am doing overlap PCR to join 5 fragents to form expression cassette and product is always trapped in the wells and is not migrating down, I did purification by PCR buffer A but still the result shows bright light up and down and and a less intense band if anyone can guide me I will be thankful..
How large is your expected product? You might want to consider a lower percentage agarose and running the gel for a longer time. You can also try loading a smaller starting volume since an "overloaded" well will have a smear.
Can you post a picture of what you are seeing? Unless your fragment is very large it should still enter the gel fairly readily.
Sure. The fragment should run through the gel irrespective of the size. And again, could be the PCR reaction did not occur and whatever you see in the gel is an actual DNA fragments.
Do your overlap PCR in to 2 time. First part join fragment 1 to 2 and in another PCR join fragment 3, 4 and 5.
Purified with a Nucleospin (Macherey Nagen) and amplify your final fragment. Use a high fidelity Taq DNA polymerase (Q5 DNA polymerase)
Try agarose gel at 1% and make a pre-inubation of the pcr product with proteinase K at 56° for 10 minutes.
Recommendations:
Use agarose with a low melting point, has lower gel strength, greater clarity, and great sieving capacity. In a well of the gel place, a control sample that you know its size to rule out flaws in your gel. The control should be displayed. To rule out failures in your PCR of interest. Also, put a positive control and use high-fidelity Taq.
Greetings
Are you trying to join all 5 fragments at once? You didn't mention the size of any. I assume the final product is quite big. Try using a range of different percentages for the agarose gel and run your product. You may find the optimum percentage of gel that will show the migration.
You can check the agarose concentration, DNA purification and amperage
In addition to all the informatively answers submitted, I want to also suggest that you should make sure you run a confirmed control negative and positive samples alongside and also start with a low melting point.
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