Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
Very explanatory video!!!! I have a question: Does it works on both TBE and TAE gels. This because Boric acid can sometimes inhibit DNA binding in Silica
Hi Georgios, thanks for your question. You can use either type of buffer, but you might see slight differences in yield. Some kits have neutralizing buffer, but it's best to check with the manufacturer if you're using a kit.
Thank you for your question. The manual for the kit that you are using should have troubleshooting tips for improving DNA yield. One tip we recommend that may help improve yield is to get as little excess gel as possible with you excise your DNA band.
I eluted my band of interest from agatose gel by using a gel extraction kit and made PCR reaction of my targeted band but after pcr I get multiple bands on gel. I want to sequence my band of interest but I am getting multiple bands after elution and PCR. Kindly suggest me what can I do in that case
Thanks for your question. If possible, you may want to run an aliquot of your extracted DNA on a new gel to confirm that you do not have contaminating DNA in your sample. If your extracted DNA looks clean, then you may need to optimize your PCR protocol. Feel free to refer to our Polymerase Chain Reaction (PCR) Protocol for more details and tips. www.addgene.org/protocols/pcr/
This can depend on how much PCR product you expect. If you don't expect a high PCR yield, you can load more sample (ex: the whole PCR reaction). If you expect a high yield, you can load less.
We recommend following the manufacturer's instructions from your gel extraction kit. For example, Qiagen's QIAquick kit recommends Incubating at 50°C for 30 min in diffusion buffer.
Thanks for the question! It's best to cut close to the DNA band, but be sure to cut around it so that you do not cut through the DNA. Cutting through the DNA will damage your sample.
Thanks for the feedback! We try to pack each video with as much information as possible, but some things just can't make it into the video. For a more in-depth look, you can check out our written protocol here: www.addgene.org/protocols/gel-purification/
The digest image was intended to be an example of a properly purified DNA fragment or insert (lane 1), as well as one that had backbone contamination from the original full plasmid (lane 2). The lower band in both samples look similar because they are DNA fragments. Sorry that this wasn't more clear!
@Athallia Qatr The commercial kits will come with an ethanol based wash buffer that will wash the salts and other contaminants from the DNA sample once it is bound to the filter column. The sample is then eluted off the column with H2O or elution buffer.
Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
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Very explanatory video!!!! I have a question: Does it works on both TBE and TAE gels. This because Boric acid can sometimes inhibit DNA binding in Silica
Hi Georgios, thanks for your question. You can use either type of buffer, but you might see slight differences in yield. Some kits have neutralizing buffer, but it's best to check with the manufacturer if you're using a kit.
Very useful. Thank you!
Thankyou for beautifully explanation
I noticed that you are using the qiaquick protocol but don’t mention adding isopropanol. Is there a reason for this?
Super explanation
Great video. This purified product can be stored as -20C overnight or should be used for ligation following the purification?
@Karina Hajdu Even at 4c should also be fine for just overnight. DNA is able to hold up pretty well.
You can indeed store the purified PCR product at -20C.
when moving the dna from gel to centrifuge tube - it said to use sterile equipment. Does this process also needs to be done under a laminar hood?
Ethidium bromide?!?! No way Jose!
Thank you❤️
Great video
How can I increase the DNA concentration? I am using this kit but still getting a very low concentration.
Thank you for your question. The manual for the kit that you are using should have troubleshooting tips for improving DNA yield. One tip we recommend that may help improve yield is to get as little excess gel as possible with you excise your DNA band.
Can you link the UV face shield that you use?
We're currently using this face shield in the lab: www.fishersci.com/shop/products/oberon-face-fit-uv-absorbing-faceshields-2/p-94229
@@addgene Thanks so much!
I eluted my band of interest from agatose gel by using a gel extraction kit and made PCR reaction of my targeted band but after pcr I get multiple bands on gel. I want to sequence my band of interest but I am getting multiple bands after elution and PCR. Kindly suggest me what can I do in that case
Thanks for your question. If possible, you may want to run an aliquot of your extracted DNA on a new gel to confirm that you do not have contaminating DNA in your sample. If your extracted DNA looks clean, then you may need to optimize your PCR protocol. Feel free to refer to our Polymerase Chain Reaction (PCR) Protocol for more details and tips. www.addgene.org/protocols/pcr/
How much volume of pcr product we have to load in a gel to perform gel purification.
This can depend on how much PCR product you expect. If you don't expect a high PCR yield, you can load more sample (ex: the whole PCR reaction). If you expect a high yield, you can load less.
@@addgene Okay i got the point and thanks for your response.
What is the temperature at heat block and time for gel to be dissolved?
We recommend following the manufacturer's instructions from your gel extraction kit. For example, Qiagen's QIAquick kit recommends Incubating at 50°C for 30 min in diffusion buffer.
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will it cut the dna if we chop up the gel containing desired band?
Thanks for the question! It's best to cut close to the DNA band, but be sure to cut around it so that you do not cut through the DNA. Cutting through the DNA will damage your sample.
add more description
Thanks for the feedback! We try to pack each video with as much information as possible, but some things just can't make it into the video. For a more in-depth look, you can check out our written protocol here: www.addgene.org/protocols/gel-purification/
Why undigested plasmid and digested plasmid have the same band?
The digest image was intended to be an example of a properly purified DNA fragment or insert (lane 1), as well as one that had backbone contamination from the original full plasmid (lane 2). The lower band in both samples look similar because they are DNA fragments. Sorry that this wasn't more clear!
@@addgene No problem. Thank you very much.
how do you wash the DNA?
@Athallia Qatr The commercial kits will come with an ethanol based wash buffer that will wash the salts and other contaminants from the DNA sample once it is bound to the filter column. The sample is then eluted off the column with H2O or elution buffer.
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ua-cam.com/video/Fv-vayq5MCA/v-deo.html (Watch this video for precautions)