4 replies to this topic

[grvsomani]

please help. i have isolated plasmid DNA (by alkaline lysis with SDS :minipreparation) without doing RNase treatment. plasmid DNA was dissolved in 20ul nuclease free water. when i run plasmid in agarose gel electrophoresis, RNA remain below 300 bp in it and plasmid DNA at 3000bp . i do overnight restriction digestion(by Hind III & Pst I), digested product should be apx. 800bp. length. but when i run gel there was no product at 800 bp. Is it necessary to do RNAse treatment before RE digestion  or enzyme concentration is required more ?
is Plasmid RNA inhibit RE digestion
Reaction of RE digestion
NFW-          up to 20 ul
10 x buffer-  2ul
enzyme-       0.8ul
plasmid-      4ul  


please help me, thanks

[protolder]

grvsomani, on 14 April 2011 - 03:05 AM, said:

please help. i have isolated plasmid DNA (by alkaline lysis with SDS :minipreparation) without doing RNase treatment. plasmid DNA was dissolved in 20ul nuclease free water. when i run plasmid in agarose gel electrophoresis, RNA remain below 300 bp in it and plasmid DNA at 3000bp plasmid digestion 13-4-2011 1.jpg . i do overnight restriction digestion(by Hind III & Pst I), digested product should be apx. 800bp. length. but when i run gel there was no product at 800 bp. Is it necessary to do RNAse treatment before RE digestion  or enzyme concentration is required more ?
is Plasmid RNA inhibit RE digestion
Reaction of RE digestion
NFW-          up to 20 ul
10 x buffer-  2ul
enzyme-       0.8ul
plasmid-      4ul  


please help me, thanks

Hola , I think that the problem is that both RE have star activities which lost the digestion site specifity specially in incubations overnight. In fact making midipreps with the traditional method without RNAse neither silica columns you can digest to check the presence of your insert. To me, making shorter time digestions you will have the whished fragment.Made a try or wait for any specialist recommendation. Buena suerte

[grvsomani]

as i was doubtful that desired insert is present in the recombinant plasmid or not?. it was confirmed by PCR using diluted plasmid as template. i got desired amplification so it prove that there is insert of accurate size in 8 out of 10 plasmid. now i am planning to digest plasmid for 2hrs tomorrow.

thanks for suggestions

[grvsomani]

i have also digested my product with Hind III, Pst I for 2 hrs incubation. both are giving smear.i have also digested with Bgl II for overnight which also giving smear in digestion.
please help me

[Shiny]

9 out of 10 times, smear after restriction digest results from contamination of initial prep. The prep can b purified using phenol chloroform. Also for the RNA present in the  prep you can add RNAse and incubate at 37c for 30min n it will get rid of all RNA.