Posted by: ericmjl | June 23, 2009

Something’s Fishy

For the past week, I have been attempting to clone an approx. 100 bp insert into our ampicillin construction plasmids (approx. 2.1 kb), and this has proved excruciatingly frustrating. After initially assuming that the transformation went fine and good (because I had obtained colonies), I digested the mini-prepped plasmids from those colonies and screened them via a restriction digest on the plasmids before running a gel of the said digests, only to find that the band sizes were not 100 bp. and 2.1 kb. Rather, I encountered a myriad of band sizes, from 300 bp to 450 bp to 1.8 kb to 2.2 kb, but none of the 100 bp and 2.1 kb sizes that I had expected.

In order to troubleshoot this, I took a backup set of ligations (which were an overnight extension of the 1 hr ligation which I had originally carried out), and did the whole motion of transforming, mini-prepping, and digesting, only to find even more bad bands. That made for a harrowing weekend of frustration in the lab. Concatamers are fairly impossible, as the EcoRI and PstI cuts don’t have compatible overhangs. A gel of a fresh set of digests + ligations yielded blank bands, which means that I just have to transform and have faith that everything goes well.

Meanwhile, the original minipreps have been sent for sequencing. I just hope that after doing a sequence alignment that everything looks okay. Then I can stop everything else and running into this brick wall.

Anyways, back to the transformation.

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Responses

  1. […] a comment » After a harrowingly frustrating weekend in the lab, I swear I will not go into the lab and do heavy-duty lab work anymore. At best […]

  2. […] carried a plasmid with little sequence homology to the original inserts. No wonder things were harrowingly frustrating. No wonder Amelia also failed to clone the lock/key/control lock in (I was expecting her to succeed […]


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