S-STEM Scholar Alaa Alshawi's Blog

This week a nother gel was run on digested lambda and plain lambda for about 4 hrs and 30 mins; after there was concern that the gel did not run long enough for the ladder to show the higher base pairs (50k to 25k base pairs). However, after running the gel for so long, the plain showed 2 bands on the gel, which was confusing because you would expect that from the digested lambda. After talking to the professor, it was concluded I might have switched the tubes for the digested and not digested lambda.  Recap on why we were experimenting with lambda DNA. An issue we are having while trying to ligate kanamycin and PGRNA is that we confirmed previously that the x-bai site is not binding at all, so to diagnose what exactly in our process is not working correctly, we are using lambda as our control (comparing it to our plasmids). We are doing everything exactly the same way to make sure we catch what we were doing wrong. 

This week we started to explore the idea of using lambda (#N3011S from New England BioLabs) as a control group to figure out what exactly is going on with the ligation of PGK, and one issue we're hoping is the problem is the digestion of the XbaI site of the fragments. One of the most important things when using this method is to do everything exactly the same way, or else we wouldn't know exactly what is going wrong.  Key information about lambda Concentration: 500 µg/ml Base Pairs: 48,502 XbaI Site: Located at 24,508 bp, leaving a 23,994 bp fragment after digestion. Lambda was digested with x bai overnight and then run on a 2% 120 ml gel for around 2 hours. 

This week we decided to gram stain our parent plate of pd-cas9 and a lab mate's plate to see if our errors are coming from that plate and if it's contaminated, and the other pd-cas9 plate looks good after gram staining; we would just use that plate.  During the week, a transformation of PDCas9 E. coli and Sonora was attempted; however, it was unsuccessful, and another plasmid extraction of PD Cas9 was performed. 

Plasmid extraction of Pd-Cas9  O.D. value is A: 5.35, B: 5.44 Only 2 uLs of ellusion buffer were used for a high concentration because it will only be needed for transformation.  Nanodop reading of PdCAS-9 E. coli  42.4 ng/µL concentrations of tube A (A260/A280: 1.84) (A260/A230: 2.99)  Tube B: 36.4 ng/µL (A260/A280: 1.85) (A260/A230: 1.66)  For transformation of Pd Cas9, only 1000 ng is needed and at least 10 µL of volume Gram stain of pd cas-9 E. coli  Contaminated so gram-stained parent plate to check if the contamination is coming from the source.  A tube of Pd cas 9 was found in the -20 freezer so passaged that on plates and created a freezback. single digested pgK with xbar, inoculated our TGY and LB plates ligation of PGRNA and kanamycin (5:1 ratio) If ligation is not successful, Lambda DNA will be used as a control to figure out exactly what we are doing wrong in the process. 

This week was mostly about gram-staining pd cas9 and learning to use the 4-way streak method for better colonies of growth on the agar plates. I learned it was best practice to streak the plate facing down for less risk of contamination, but I prefer to streak face up for more accuracy.      When gram-staining growth from these plates, in this case Pd Cas9, it's good to know that a little more time on the ethanol step won't harm the quality of the stain too much, especially if the cells are compacted.  After these plates were gramstained, they were passaged onto R2A and TGY plates poured during the week. 

We started the week by doing a plasmid extraction. We still need to confirm the results with a gel. pgK Gel. During these next few weeks, plasmid extractions have all been coming out with bad nanodrop results, which left us on a standstill and confused as to what could possibly be going wrong with the extractions.  still haven’t figured out what went wrong with the previous gel for pgK. Might have used the wrong ladder, which threw everything off. This highlights how important it is to label everything properly and write things down.