Week 3: Lab Work

This week I focused on lab work at Cold Spring Harbor Laboratory (CSHL).

At the moment I am 1/4 the way through my research at CSHL, right now my mentor, Dr. Lindsay, and I are focusing on checking the goal of my main experiments this week is to double-check that things are going well. To do that we are working on genotyping our Arabidopsis to make sure they mutated and to check on cloning

On Tuesday, the lab went to listen to one of their colleagues in present their current work, then we went to work. I made a multi-site gateway LR reaction. Gateway cloning is an efficient and effective way to insert or excise genetic material (i.e. recombination) in vitro (in a lab environment) [1, 2, 3]. A multi-site gateway LR reaction is used when inserting multiple DNA fragments. This reaction uses LR clonase to insert DNA fragments into the bacteria/ cells that we want to transform (i.e. introduce foreign DNA). After that, Dr. Lindsay, her mentee, Pan, and I went to the lab’s farm. At the farm, I transplanted the transformed Arabidopsis, that I had plated two weeks ago, into the dirt.

^The plate with white and green plants is the group of plants that weren’t screened for transformed plants before plating, instead, the transformed plants were selected for by an antibiotic. The plants that were transformed are alive and green, the ones that are white were not transformed and are dead. The plate with fewer plants is all transformed plants that were selected before planting. The goo on the other plate is agrobacterium (check out my week 1 post to learn more about agrobacterium).

After transplanting the baby Arabidopsis, I went to the growth chamber with Dr. Lindsay to check up on our previously transplanted plants (see below). We were happy to see that based on the phenotype (physical characteristic) there may be two mutants! We then collected a small leaf from each plant for genotyping.

On Wednesday, I used the leaf samples that we collected to try out a simple DNA extraction protocol. The protocol called for me to crush and combine the leaves with the buffer then boil and dilute the solution then use it as the DNA template for a PCR reaction. Lastly, I ran the results on a gel and found that this method was unsuccessful. My mentor and I plan on trying a different procedure next week.

On Wednesday, I also used my LR reaction to transform some Mach-1 E. coli cells. These E. coli are named Mach-1 for the speed at which they grow. After the transformation process I plated the cells on an LB-Spec plate, LB is just the media type and spec. is short for spectinomycin, an antibiotic that is used for selection and preventing contamination. On Thursday, my mentor collected some of the E. coli from the plate I set up on Wednesday and put it in a solution.

On Friday, there was a lab meeting in the morning and then a big presentation. After everyone went back to the lab and got to work. I used the solution my mentor prepared to perform a restriction digest (used to cleave DNA at specific sites can be used for multiple reasons, I was using it to confirm that the genes that I had hoped to insert were there or not). My first step for this was to do a mini-prep to prepare the plasmid (a circular piece of DNA usually found in bacteria, in this case referring to the transformed DNA from the LR reaction), then to use the plasmid for the restriction digest. To check if the cloning worked I ran the restriction on a gel. However, the gel showed that the fragments were the wrong size and that there was probably an issue with cloning. This is quite common. In fact, cloning is notorious for causing a lot of tears for scientists, so I see this as a momentary hiccup.

^The gel for the restriction digest

This week I was able to work in the lab more independently. While still needing some clarifications for different protocols, I have become more confident in the lab.  

Sources:

1. P., A. (n.d.). Recombination: Definition, Mechanism and Types | Microbiology. Biology Discussion. Retrieved April 18, 2022, from https://www.biologydiscussion.com/genetics/recombination/recombination-definition-mechanism-and-types-microbiology/65220
2. Reece-Hoyes, J. S., & Walhout, A. (2018). Gateway Recombinational Cloning. Cold Spring Harbor protocols, 2018(1), pdb.top094912. https://doi.org/10.1101/pdb.top094912
3. Soriano, M. (n.d.). Plasmids 101: Gateway cloning. Addgene blog. Retrieved April 18, 2022, from https://blog.addgene.org/plasmids-101-gateway-cloning