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.


Nia_Fernandez said...

what do you mean by some of them have both?

those bands do look good!

Brad Hall said...

Nice data. Exactly what we were hoping for. However, no one in the lab really has an idea of how to tell this is exactly what we were looking for...why did you do this? Since this has been up for a while, I figure you aren't updating it. Therefore, I would like you to update it with the following information.

Please explain why you did this so that others who read it can be brought up to speed. How does this gel differ from a normal cycle course? Explain the PCR gel (results) and they why (discussion). What do the bands mean? What size are they? How can you say some have inserts? What about the gel tells you this? Which wells/colony numbers correspond to the bands? Is there anything discerning about the "insert" bands, which correspond to colonies, that could help you differentiate colonies (size/color)? This may help you pick colonies to do a large scale clone check for sequencing.

You mentioned there were a few blue colonies and white colonies. When explaining data, don't say "Here's how the plates looked". Rather say something like, "The cloned colonies were grown on LB+Amp plates shown in Figure 1". Explain what the top/bottom/left and right plates are. Are they different rounds, different cloning reactions of the same round? What does the A and B mean? For the white colonies, if they were satellites, it wasn't the lack of x-gal that made them that way. Think about could you spto x-gal into specific places with the coli rollers. Can you think of why else there would be these colonies? What did I tell you about the age of the plates (made in late July)? How will this affect the antibiotic? Why are the white colonies smaller? Did they start growing later? Maybe after some event?

You will likely need to actually label your data using some version of figure editing software (Powerpoint, Illustrator, InkScape, etc) to be able to explain it. Don't just post primary data, but instead post data that has labels on it so you can describe the results better.

Emma Weiss said...


Is there a difference between plates? Brad I'm confused by all your questions! You lost me by the third one. We should have an office hour for COMPUTER APPLICATION help to teach us how to present our data in a presentable manor.

Brad Hall said...

Katherine: You answered most of my questions...great job. The post is much more clear! Two clarification points you may decide to update
1.) - the cells don't actually grow blue as you described. They grow and produce a functional B-galactosidease which cleaves the X-gal releasing a blue chromophore.
2.) the small white sattelite colonies don't actually have an insert. Remember back from the cloning lesson from the spring that the satallite colonies are smaller because they couldn't start growing until the ampicillin was inactived (chewed up) by the beta-lactamase found in plasmids of surrounding bacteria (the large blue/white ones). Ampicillin is bacteria-static at a certain concentration which means when it's there at sufficiently high concentrations it doesn't kill the bacteria but just prevents them from growing. Therefore, when the surrounding cells with plasmids (regardless of insert) produce b-lac, the enzyme + relatively old ampicillin will fall below the "static" threshold and the non-cloned cells will begin to grow.

Emma, Katherine updated her original post which may be why you don't understand my questions...Katherine updated her post to address much of it. But you make a good point about "office hours". I am in lab on Mondays from 10a-1p and 3p to 5p. You are free to stop by and chat. Or POST QUESTIONS TO THE BLOG!!! Either way, I am going to have you post the question and your interpretation of my answer to the blog eventually ;)

Alec Rezigh said...

We are learning about this experiment in my Genetics class. Although I remember the general procedure, this was a great refresher. Thanks Katherine, and best of luck!