Hi
I am trying to set up the reactions with my plasmid, insert and RE. Usually the standard final volume is 20 uL or 50 uL (it depends on the protocol provided by the manufacturer) with 1 ug of DNA. My plasmid is concentrated 10 ug/mL, so if I want to use 1 ug of DNA I should take 100 uL from my stock. I wonder if I should rearrange all the volumes of the other reagents (buffer and water) to readjust them to this volume of plasmid used. If so, I wonder how it would be possible to switch with bigger volumes to a ligation reaction. I specify that my protocol does not include a gel-purification step. So, will I have to purify with some kit or simply precipitate the cutted plasmid, before proceeding with the ligation step, in order to resuspend it in more suitable volumes?
Thank you for your help
Dear Laura
in principle you can scale up the volumes just maintaning the proportion beetween the different reagents however is possible that for purification you have to split your reaction in more than 1 coloumn.
However i suggest you to evaluate more modern cloning approaches as the PIPE cloning which do not require to pass through the digestion step which is often a tricky step.
if you are interesting you can find more infromation about PIPE in the following link of my blog
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
as well as in the following papers:
https://pubmed.ncbi.nlm.nih.gov/18004753/
https://pubmed.ncbi.nlm.nih.gov/18988020/
best
Manuele
the rules for DNA digestion are 1/not more than 0.1ug/ul of DNA and 2/not more than 10% (final volume ) of enzyme (more than 5% of glycerol (from the enzyme can interfers with the enzyme accuracy) 3/ unit enzyme concentration depends on the definition by the supplier (generally 1u/ug of DNA). However the rule for DNA concentration if very flexible (more dilute is not a problem) and with the quality of the restriction enzyme you can-over digest to x10 more enzymes...
if your plasmid DNA is only 100ng/ul I would try to get a better prep (is it a very lage plasmid with low copy replication origin?) or reprecipitate and concentrate the plasmid...
Try precipitating your plasmid with LiCl and ethanol and use less water to resuspend it. Scaling up is for more ug of DNA
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