Week 9: Presenting my findings at CSHL

Welcome back to my blog!

My presentation for the Jackson Lab is next Friday, so this week I have been prioritizing wrapping up my work at CSHL. So far, this has entailed reviewing and organizing my lab notes and picking out images to share my results. In addition, I have been doing some experiments and organization in the lab and at the farm.

On Tuesday, I purified the PCR product from last week’s PCR (see gel below).

The picture above is the PCR product on a gel. The bands/horizontal lines indicate the size of the DNA segment that I amplified via PCR. The further down the band the smaller the fragment. The first two rows are the 23+59 CRISPR transformed plants and the two rows below are the 21+77 CRISPR transformed plants. The 21+77 PCR product is faint but you can see that most of the bands look uniform, on the other hand, the 23+59 PCR product has a few inconsistencies. (The samples with stars above their band have irregular bands.) The variety of band sizes and the number of bands among the 23+59 PCR products suggests that there were successful edits, while the uniformity of the 21+77 PCR products suggests the opposite. As such, I only purified the PCR product from our 23+59 CRISPR transformed plants except for the starred samples. (The reason why we don’t want to sequence our multi-band samples is that the sequencing data that comes back is usually chaotic and not informative.) After purifying the PCR product, I prepared the samples for sequencing.

On Wednesday we went to the farm to do some maintenance for our plants. First, we planted some N. Benth, in case anyone needs it for experiments or demonstrations. In addition, we transplanted the last batch of N. Benth seedlings into individual containers. After that, we staked our T1 Arabidopsis plants so that they do not grow into each other and recorded the IDs of which plants looked different (i.e smaller or the ones that had more branching). After that, I collected seeds from some older T1 Arabidopsis plants individually. After that, my mentor and I went back to the lab to discuss my presentation outline and what preparations I need to do.

On Friday, after the lab meeting, my mentor and I worked on analyzing the sequencing data from the purified PCR product we sent in on Tuesday. As I mentioned above, if we sequence the samples have multiple bands as is, the results will not be very informative. So, my mentor and I decided to try a gel extraction to select the individual bands to sequence.To perform the gel extraction I first made a gel and ran 3 of the samples with multiple bands. When the gel was done my mentor and I worked on cutting the gel to collect the individual bands for sequencing. After that, I went through the process of purifying the PCR product and extracting it from the gel. After purifying the gel I prepared the samples for sequencing. I also ran a PCR of a few of the samples with multiple bands, so I could more clearly see the size of the bands.