how to extract plasmid spotted on paper? - (Jul/27/2007 )

Hi All
I got the plasmid carrying my gene of interest spotted on a paper. To elute the spotted DNA, the filter paper was soaked with 100 ul TE buffer for few hours at room temperature and centrifuged, and the extract was used for transformation. I tried following variations for transformation and screening of transformants:

both ‘home made’ and ‘commercial’ ( MAX Efficiency, Invitrogen# 18258-012) DH5a competent cells and BL21(DE3) pLysS were tried for transformation: volume of extracted DNA sample (obtained above) was varied from 1-25 ul for transformation in different sets of experiments: 100 ug/ml Amp and 50ug/ml Carbinicellin were tried separately to screen transformant ( plasmid is derivative of pET 20b+ which is ampR).

Unfortunately, I am unable to transform the cells, although the positive controls are consistently giving good transformation indicating the cell preparation and transformation protocol are fine.

I believe, I am unable to extract the spotted DNA (0.5ug) completely.

I will highly appreciate if anybody gives me some suggestion to solve this issue.

Thnanks

just put 100µl of miliQ water on the paper, and make it rehydrate by 37° 10'.
Then pick 10µl for a electroporation or heat shock transformation.
Goes all good.

but maybe ask the person who send it to you? there may be particular issues for that plasmid.
Otherwise, reask for sending.

where did you get the plasmid from? A colleague of mine recently had the same problem as you are facing, however when he plated some on plates with kanamycin, colonies appeared. Apparently the wrong plasmid was sent, but at least then you know your transformation is okay.

Thanks for the messages. I did asked for plasmid for second time and which I got. And second time also it was the same result (after trying all combinations). i dont know what to do.

try to warm up the filter paper and water or TE upto 65C for a few minutes and then retransform it.

I use the following method for extracting DNA spotted to thesis paper:
-Use a micropunch (2mm diameter) to remove the portion of the paper containing the DNA from the excess
-Soak in 20 uL TE for 15 minutes
-Add 5 uL of the extracted DNA to 50 uL chemically competent cells

Even though your positive controls have been working you may want to try extending your heat shock. That was my major problem when I started working on a DNA spotting protocol. I heat shock at 43 degrees Celsius for 1 minute.

'Sam~'


Can you explain me the science behind heat shock?


Thanks.

During the heat shock the e. coli cells take up the plasmid from the surrounding fluid. I believe the extreme heat makes the cell membrane more fluid, allowing the plasmid to pass through it, but it may be different or more complicated than that.