For cloning purposes, I have to double digest PCR products and plasmids 20 ug of each. Standard 50 uL restriction digestion reactions can accommodate up to 1 ug of DNA. According to some experts, it is possible to digest 10 ug in a single reaction. Can someone advise on the recipe, as I have never prepared it before?
10ug used to be routine when Southern blots were commonly used. The unit of activity of a restriction enzyme is given by 1 unit of restriction enzyme will completely digest 1 μg of substrate DNA in a 50 μl reaction in 60 minutes. A 50 ul reaction volume will be fine and theoretically 10u of enzyme are needed for 10ug of dna cut for 1 hour but many researchers will add 20u of enzyme and cut overnight to ensure that there is complete cutting with few partials. Then take a small amount ( 3ul) and run a checker agarose gel to check for a smear of cut dna that indicates good digestion.
The definition of activity is based on average spacing between cut sites in genomic dna but when cutting pcr product and plasmids and with rare cutters there are many more cut sites than expected compared with genomic dna so you should use more enzyme and cut for longer to ensure good digestion
If you need to double digest 20 μg of PCR products and plasmids in a single restriction digestion reaction, you can scale up the reaction volume to accommodate the larger amount of DNA. Here's an example recipe for a larger-scale digestion reaction:
It's important to note that large-scale digestion reactions may require longer incubation times or different reaction conditions compared to standard-scale reactions. Ensure that you follow the manufacturer's guidelines for the specific enzymes you are using.
After the digestion is complete, you can proceed with downstream applications such as cloning or further purification steps. Keep in mind that large-scale digestion reactions may introduce additional challenges, such as increased DNA loss during purification, so optimization and careful handling of the larger reaction volumes are necessary.
best.
Hi Md. Amzad Hossain
I would suggest you to do 20ug in a single reaction.
Mix:
20ug DNA: x uL (calculate as per your stock)
10X Reaction Buffer: 20uL
Enzyme (1U/uL is generally available): 20uL
Nuclease Free Water: upto 200uL
Hope it helps,
Subham.
the rules for digestion are
not more than 0.2 microg/microl DNA (final)
not more than 10% volume of enzyme (over 10% glycerol you may have some unspecific cuts)
1u of enzyme /ug of DNA (with good enzymes now you can use 10x more if you want)
for 1h at 37°C (exept some enzyme cleave at 65°C ; to be checked)
use the buffer recommended by the provider
same for double digestion: check if the buffer of the two enzymes are compatible; if not start with the buffer with less salt then after inactivation of the first enzyme (10min 65°C generally) adjust the salt and volume and add the second enzyme
now with the high quality of restriction enzymes the rules are flexible (more enzyme, longer time (in that case the pb will be the quality of your DNA not your enzyme) and some provider have a "universal" buffer usable for a lot of enzymes ....
Hi Amzad Hossain.
I am expecting that you wants to digest 20ug of DNA and PCR product each.
Let's discuss some recipes. I am sure this will help you a lot.
Your final volume should be upto 300ul for this.
1. Use 20ug/200 ul of each DNA and PCR product.
2. 10x buffer 30ul.
3. 1U/ul of atleast 3ul restriction enzyme and then finally make upto 300ul with nuclease free water fresh double distilled autoclaved water.
Thankyou.
JUNAID.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Condensed Matter Physics
- Soft Matter
- Liquids