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.

  We talked about how we’d already digested kana with XbaI before and planned to ligate it and do PCR. We also went over how PCR works: PCR is targeted replication. The machine heats up and breaks the hydrogen bonds, so the DNA separates into single strands. The strands keep separating. Primers go in at their kneeling temperature, which for us is 57°C. This tells the polymerase where to start. At 72°C, the polymerase copies the DNA, going from one strand to two. The machine repeats the process for a set time. Also… pdCas9 is 6,700 bps. Deinococcus usually grows between 30°C and 37°C, but Sonora likes 30°C. E. coli grows best at 37°C. ran a gel to check pgK again, but the results didn’t make sense, and we couldn’t figure out what went wrong.

LB Broth Preparation and Transformation: LB broth was made for the growth and transformation of E. coli . Overnight Ligation: Ligation of kanamycin and pgRNA was planned for overnight because of past unsuccessful ligations. Quick Ligation Protocol: Quick Ligation Buffer: 10 µl used to maintain pH, preventing the ligase from degrading pgRNA. Kanamycin Calculation: Based on the online calculator, 177.8 ng of kanamycin was needed, based on a 5:1 ratio. pgRNA: 2 µl of 50 ng/µl concentration was used for a total of 100 ng. Kanamycin: 3.5 µl of 50 ng/µl concentration was used for a total of 177.8 ng.

This week was focused more on the transformation of Kanamycin and PGRNA. kanamycin cleanup  NEB monarch cleanup kit  added 260 µL of binding buffer to 52 µL of sample (5:1 ratio)  15 ul of ellusion The nanodrop read 74.1 ng/µL with an A260/A280 concentration of 1.84 and an A260/A230 concentration of 1.13. Run gel on kana after cleanup. 900 base pairs   double digest after cleanup  After digesting kanamycin and PGRNA, set up ligation.  If ligation looks good, need to immediately place in E. coli. gram stain PGRNA E. coli transformation of pdcas-9  2 tubes, one with competent cells and a control tube with no competent cells  If transformation was successful, cells should be resistant to ampicillin.  Sonora D. is resistant to tetracycline. A plasmid is a small piece of DNA with essential genes like antibiotic resistance.  PCR polymer chain reaction  give templates for primers to replicate  

Gram-stained Sonora plates from 9/13 short rods  Gram Positive plasmid extraction of Cas9  Gel excision prep cut wanted band from gel  For every actual weight of agarose gel, add 100 µL of binding buffer.  Place in incubator until gel is dissolved (should be yellow if buffer is working).  Transfer 400 µL of gel solution to a microcentrifuge tube for 1 min, then discard the flowthrough  100 µL of binding buffer for 1 min, then discard flowthrough.  100 µL of wash buffer, then centrifuge for 1 min to discard flowthrough Then place the column in a new centrifuge tube to add 20 µL of elution buffer. learned that E. coli is what is used to keep bacteria restriction sites of PGRNA is about 6 bp cut a site to make linear  C1. V1 = C2. V2  C 1 starting  concentration  V1, starting volume  C2, ending concentration  V2 ending volume 

During this week I was shadowing my current lab group:  Cut  Kanamycin from its X-bai site and ligate it to PGRNA.  learned that Sonora dionecoccus is a bacteria that is highly resistant to UV light.  gram-stained Cas-9, however, they looked contaminated.  learned that competent cells are cells that can easily take in foreign DNA, making them good for transformation. Performed a transformation of Deinococcus radiodurans Thaw competent cells.  Aliquot 100 µL of competent cells into a microcentrifuge tube.  Add 1 µL of plasmid solution to a microcentrifuge tube with competent cells.  place on ice for 15 mins Place in incubator at 37°C for 45 mins. agitate the tube every 15 minutes  Transfer contents into a 15 ml centrifuge tube with 4 ml of TGY broth.  Incubate in a shaking incubator for 16 hours (overnight).  after 16 hours, plate onto R2A agar plates with antiobiotic for 4 day Made TGY agar plates with ampicillin for D. Sonora....

 The first few weeks of Lab consisted of mostly shadowing groups and taking a lot of notes about the nature of the lab and what kind of experiments are run during the week. Below is a list of things I learned or observed  How an agarose gel is made for an electrophoresis machine which one of its uses is to run PCR products on to determine if transformation or ligation has occurred based on base pairs How to load a gel and the procedure for preparing products for the gel like how much dye and how much PCR water needs to be used in order for the bands created to be visible  Also on the first day I observed how some procedures for plasmid extraction is to cut out the band from the gel after it has ran on the electrophoresis machine. later on during lab I learned that using this technique might not be the best to use if you have a small amount because you lose around half of your sample or product  E.coli grows in the 37 degrees Celsius incubator.  How to make a con...