Cloning and Sequencing of 1027 selection Part 1

I ligated and cloned the sequences (hopefully). Here's how the plates looked:


The later rounds have an alphabet after them (A/B) because during the summer, I split my total reaction into two selection rounds in the hopes of finding different aptamers. Both selections are done with the same parameters. The plates were ligated, cloned, and plated with the same parameters also. All four plates show large blue colonies surrounding by smaller white colonies with larger white colonies on the periphery of the satellite colonies.

The plates were treated with X-gal and Ampicillin, the former to track which cells had the Plasmid + insert and the latter to keep cells without the plasmid from growing. They are basically “weeding out” techniques that cut down sorting time. With the plasmid + insert, the colonies should grow white on an plate with X-gal and Ampicillin, but with only the plasmid, the colonies will grow a bright blue. The smaller white colonies could be Plasmid+ insert colonies, or they could be satellite colonies which are actually blue colonies in disguise around the larger blue colony which has absorbed all of the nutrients and materials (X-gal, ampicillin, ect) from the surrounding areas. The blue colonies are blue because they have taken up a plasmid without an insert and thus the lacZ gene is not disrupted so they are able to make B-Galactoside. Since the plates were made in July, it could affect how well the ampicillin works and since the X-gal was rolled on with colli rollers, the white splatters of colonies could be the effect of uneven spreading. The white colonies are smaller, perhaps, because with the insert, the genome is now larger and takes longer to copy.



This gel was run to test whether or not the white colonies had inserts in the plasmids since they were quite small and looked like they could be satellite colonies from the large, blue colonies. The results show that although there are multiple bands for some of the colonies, there are inserts at the 300BP mark, which is where the plasmid + insert should be. The colonies were randomly chosen and rubbed with a pipette tip to gather cells for the PCR reaction. Some of them show no insert at all which could be that they grew only after the large colony had eaten all of the ampicillin.