Dear Friends,
I am trouble shooting since a long for isolation of my cloned construct in the strain E.oli DH-5 alpha.
I have tried Qiagen kit but yield is very low to proceed further any work (isolating in 15 micro litre and concentration comes around 30-60ng/micro litre by kit)
Recently I have followed manual isolation method and through the end everything looks positive including the absorbance value by nano drop which shows good amount around(1-2 micro gm/ microlitre) with 260/280 value around 1.8-1.9 , RNAase treatment is also done before nanodrop observation too,
method link
https://msu.edu/course/css/451/LabProtocols/Plasmid%20Isolation%20Using%20Alkaline%20Lysis%20(Exp%203,%20CSS451).pdf
But surprisingly whenever I attempted to look on the gel, nothing is observed, completely blank (neither smear nor any band ) parallel I loaded my low amount (on the same gel) kit isolated plasmid which shows band (loaded amount 200-250ng) but not manual method shows anything that seems me as false positive reading on nanodrop with a good peak of nucleic acid on nanodrop.
Can any one suggest me to get my good amount of plasmid with positive observation on gel
Dear Amber
regarding the results of the kit, can you provide to me some more details about vector and protocoll used for culture prior plasmid extraction? eg. media, amount of antibiotic, time of growth.
When you refer to Qiagen Kit, did you mean MINI KIT?
First of all i would like to suggest to you the simples solution that is change the kit and try the Omega Biotek MINI KITS, starting from 10ml of LB culture which is my experience perform much more better than QIagen and other kits.
You can find an example of this at PAGE6 in my blog: PRoteoCool https://proteocool.blogspot.com/
regarding the manual purification, it is difficult to know where was the problem but from your results I(very high concentration at nanodrop but not signal on agarose) it seems that you have DNA or RNA small fragments that assorb at nanodrop but are too small to be visualized in agarose gel.
good luck
Manuele
Yes
It is mini prep kit
Usually i take 5 ml culture inoculation
Antibiotic ampicillin i use around 50 to 100 micro gm /ml concentration
Vector is pAbAi vector from yeast one hybrid provided by clonetech company
Although it is mentioned low copy but previously i got good amount when i cloned in Ecoli TB 1 strain
Media is LB BROTH
Time of growth around 16 to 17 hours.
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