Need some help here. I am trying to verify if the band on my PCR gel is correct or not. What is the fastest/easiest way to do it? Please advise.
While it is true that direct sequencing of the PCR product is the most accurate way to verify, there is a much simpler and faster way. Assuming you know the sequence of your presumed product, design another set of PCR primers that are completely internal and would generate a smaller but defined product size. Purify the first product and then use the second set of primers to see if you get the predicted band size. You can get your confirmation the same day.
One thing you have to check the by running the gel and find the size of the band length and after that if you are confused then go for digestion of the sample to confirm about the band length .
if you have a specific band after running your sample on the gel you can clean your PCR product using a cleaning kit and then you send the clean PCR product for sequencing with your specific primers. if you have more than one band on the gel . you cut the band on the proper size (your band expected size) then clean it and send it for sequencing with the spicific primers
You must remove all residual PCR primers and unincorporated nucleotides by specific kit
Just amplify your target DNA fragment by PCR, then check your PCR product for its band size and quality and sequence that PCR product using forward/reverse primer which are used for amplification.
Hi, Jason. One thing you could do, taking advantage of the fact that you know your sequence is the usage of restriction enzymes. Check your sequence and look for some specific restrictions sites that only your gene of interest has. (Try using this site, http://tools.neb.com/NEBcutter2/). Do the digestion, run the gel and see if your predicted subproducts appears, if it goes well, you could assume that is you obtained your product.
Doing this or going for Sanger sequencing, you will need to purify your products (There are several ways and kit to do it, so don't worry). The macrogen service offers you the chance to send the samples unpurificated, it cost 1 dolar more, but it helps a lot if you don't have the time to buy and await the arrival of it.
If you need anything else, don't doubt on writing me.
You can use PCR product for sequencing after clean-up (you need to be sure there are no byproducts) or after cutting out specific band from gel (more safe). The most important thing is you need to use proof-reading polymerase for PCR.
While it is true that direct sequencing of the PCR product is the most accurate way to verify, there is a much simpler and faster way. Assuming you know the sequence of your presumed product, design another set of PCR primers that are completely internal and would generate a smaller but defined product size. Purify the first product and then use the second set of primers to see if you get the predicted band size. You can get your confirmation the same day.
In my experience, which is limited to PCR fragments amplified from bacteria DNA templates, sequencing is simple. Simply perform a 50 ul amplification reaction and then purify the reaction products with a standard bind-wash-elute spin column. There are many on the market, but I generally use the Roche High Pure PCR Product Purification Kit. The entire clean up takes less than half an hour, and usually yields 2-7 ug of DNA dissolved in 100 ul of buffer. You can submit about a microgram of the DNA for sequencing, using the same primers that were used for amplification. Unless the PCR reactions is very poor, with many artifacts, there is no need to cut bands out of gels. The purified PCR products are clean enough to sequence. I've submitted hundreds of sequences to GenBank and closed the gaps to finish a genome sequence using this very simple approach.
Sent the pcr reaction to Macrogen they Will do the clean and sequencing for less the. 10 usd
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