I am going to cut one gene polymorphism but it does not work. If you know, please inform me of the formula to calculate the amount of this enzyme.
You must use 5 ul (minimum) enzyme for 1ug DNA and incubation time is minimum 1 hour. If you use fermentas fastdigest enzyme you can use 1 ul for 1 µg of genomic DNA in 30 min, or 5 µg of genomic DNA in 3 hours.
Hi Reza
I'll suggest you use the protocol given by the company from which you have purchased NdeI
bye
you can use protocol described by the fermentase company, instead you can make several optimation using several parameters, like time for digesting, DNA concentration, and enzymes unit if the protocol from the manufacturer doesn't work properly
best regard
hi thanks to all my friends,
I am checking 0.05 re 1.6 buffer 14.8 H2O and 8.5 pcr product but it is not working
I have another question is there any thing to use as a buffer when RE buffer is finished?
regards
are you sure that the sequence recognised by this enzyme is present in your PCR product or fragment? It has 7 recognition sites in lambda. When you fail to digets lambda DNA then you can say its not working
I would suggest you to use 0.5micrograms of your PCR product and cut it by using 10 units of the enzyme in a total reaction volume of 30 microlitres for 3 to 5 hours. If the enzyme is not mentioned as STAR activity then you can try longer digestion period. BUT the most important thing is that the DNA/PCR product you are putting should be free from any irrelevant reagents (use purified DNA). You can prepare the buffer yourself as per the formula given in the company's leaflet.
I would recommend to use fermentas FastDigest enzymes if available, they work efficiently and quick. Complete digests in 5-10mins.
Usually 1 unit is defined as the amount of enzyme necessary to digest 1microgram of a target DNA (usually phage or a plasmid) in 1hr at the optimal temperature (often 37C). NdeI is usually sold at 10 units per microliter, so you should be able to use 0.5microliter and digest your PCR fragment in few minutes. If you don't get digestion, then the 2 main possibilities are: there is no NdeI site in your fragment or your enzyme is completely down.
there is still the third possibility that the NdeI site is not accessible to the enzyme (it happens in some plasmid, where a site is present by sequencing but is not cut by the corresponding enzyme, while other sites elsewhere for the same enzymes get cut normally). If you can get your fragment to be sequenced you can check if the site is there. Or you can borrow NdeI from another lab and do a digest
Go to Fermentas web page in the enzyme description there are recommended protocols and also plasmids listed that are cut by the enzyme (e.g. pBR322, pUC18/19) and include digestion controls to your experiment (one with and another without enzyme) this way you will be sure is your enzyme work.
you may also have not enough purified DNA preparation and some inhibitors that block digestion (using TE as a buffer for DNA use 10x diluted one, because EDTA sequester Mg2+ and inhibits enzymes)
Are you sure that the polymorphism exists among the population studied? Check the frequency of the polymorphism.
Hi,
There can be several reasons of the problem. The PCR product has to be cleaned up in order to remove the PCR buffer and digest it after this step. If the NdeI site is at the end of the PCR product you have to be sure that there are at least 6 basis overhanging sequence. The enzyme might old, use control DNA to be sure it is still OK.
Best regards,
Istvan
Alternatively, you can try the following formula for calculating the number of restriction enzymes:
For example, you need to cut 1mkg PCR product length 1,6 kb by NdeI restriction enzyme in one hour. The restriction enzyme in the PCR product has 2 restriction sites.
Phage lambda have 7 sites for the restriction endonuclease. Ie one site accounts for 6.9 kb (48/7 = 6.9).
In the PCR product 2 restriction sites and length 1,6 kb, one site will be in the 0,8 kb (1,6 / 2 = 0.8).
Then, in comparison with the phage, restriction sites in the PCR product are 8.6 times frequently (6.9 / 0.8 = 8.6), respectively, and the restriction enzyme required 8.6 times more, ie 8.6 units.
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