11 November 2019 2 8K Report

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

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