This week, I did molecular cloning for the first time! It was a great learning experience.

Monday

Today was focused primarily on reading and poster design. The poster is still a work in progress, but we got valuable results this week that will be going on the poster!

Tuesday

Today, me and Clark began the three-day molecular cloning process to test its functionality. To set up the reaction, we added 10 microliters of 5X Q5 reaction buffer, 1 microliter of 10 mM dNTPs, 2.5 microliters of 10 micro-molar forward primer, 2.5 microliters of 10 micro-molar reverse primer, 1 microliter of template DNA (specially designed), 0.5 microliters of Q5 high-fidelity DNA polymerase, and filled up the remainder of the tube with nuclease-free milli-Q water. This reaction was gently mixed and the liquid was all collected to the  bottom with a quick centrifuge spin. The PCR tubes were then transferred to a PCR machine and the thermocycling procedure began. Initial denaturation was at 98 degrees Celsius for 30 seconds, then 35 cycles were run, and the final extension was at 72 degrees Celsius for 2 minutes. Finally, it was held at 10 degrees Celsius infinitely, then stopped on the next day.

This was a very lengthy procedure. I want to go ahead and explain the principle behind PCR. PCR is the abbreviated form of polymerase chain reaction, which is a laboratory technique for rapidly producing, or amplifying, millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail through agarose gels (which is what we did in our experiments). The PCR process has six steps: initialization, denaturation, annealing, elongation/extension, repeated cycles, final elongation, and final hold. Before I explain the steps, I’d like to explain the components that go into PCR. There are five main ingredients needed for PCR. Polymerases are the first ones; they are enzymes that, when subjected to ambient conditions, are able to assemble new strands of DNA from template DNA and nucleotides. The next component is the template DNA. This is the DNA that the polymerase (typically Taq) will read and copy; this DNA can be genomic, plasmid, or cDNA, and in our experimentation, it is plasmid DNA. Next, PCR requires primers, which are short fragments of synthesized DNA that bind to your specific template DNA. These have to be specially designed, we designed and ordered ours last Friday. The forward primer designates the start of PCR and its sequence is the same as the 5′-3′ template DNA sequence. The reverse primer designates the end of PCR, and its sequence is the reverse complement of the template DNA. Next, you need nucleotides. As the monomers of DNA, nucleotides are necessary for making copies of DNA. For most DNA PCRs, deoxynucleotide triphosphates (dNTPs). Finally, you need buffers. For the purposes of our research, we utilized the Q5 buffer, which helps optimize DNA denaturing, renaturing, and polymerase activity.

Next, I’d like to explain thermocycling. The above ingredients are added to a PCR tube, and the tube is thermo-cycled. The first step is initialization, in which the reaction is heated to 98 degrees in order to activate hot-start polymerases to denature the template DNA. Next is denaturation, in which the DNA and primers are denatured to be able to efficiently and effectively anneal to each other in the next step, which is annealing. In annealing, the reaction’s temperature is rapidly lowered so that the denatured primers can form Watson-Crick base pairs with the template DNA. However, the temperature is not significantly lowered as it still must be high enough that only the most stable, perfectly paired double-stranded DNA structures can form. Usually, the perfect annealing temperature is only a few degrees lower than the melting temperature of the primer pair. Moreover, during the annealing step, the polymerase will bind to the primer/template DNA complex, although it will not start reading until the temperature is raised in the next step. Following annealing, elongation/extension is done and repeated 35 times. The reaction is rapidly heated to approximately 72 degrees, and this is when the polymerase begins reading in the 5′-3′ direction and copying the template DNA in the 3′-5′ direction. The higher temperature during this step reduces non-specific primer/template DNA interactions, which aids in increasing the specificity of the reaction. After steps 2-4 are repeated 35 times, the final elongation is done. The reaction is held at 72 degrees for several minutes to allow polymerases to finish reading whatever strand they are currently on. This step, although optional, can help reduce the number of truncated copies in the final product. The last step is the final hold. This is necessary because, as PCR can often take a few hours, they are often done overnight or when the researcher has stepped away. In this step, the PCR product is held at 4 degrees until the tube is taken out.

Wednesday

On Wednesday, the work started immediately at 8:30 AM. In the morning, we created a 1% agarose gel (a procedure I had already done in previous lab experience) and verified that the overnight PCR had worked. To do this, we ran a molecular ladder as well as the PCR sample through the gel and analyzed the band. Once we were sure the PCR had succeeded (as the band was present), we discarded the gel and moved on to the next steps.

Since we had received some fresh PVDF paper, we switched gears and ran an SDS-PAGE gel and Western blot with the same samples from last week–these are the ones that did not work on the Western blot due to the different type of paper used. Once this Western blot was set up for the 9337 samples, we did the regular washes with 10% milk and primary antibody, concluding this process at the end of Wednesday by putting it on the shaker in the environmental cold room.

Next, we did blunt-end ligation cloning. The blunt-end ligation is a non-directional cloning method, meaning the insert can ligate to the vector in two possible directions. For protein expression, the insert must be in one particular direction, and usually, the desired directional clone can be identified using PCR. In blunt-end cloning, both the vector and the insert contain blunt-ends. During the transient association of the ends of the vector and the insert, the DNA ligase enzyme seals the gaps. The T4 DNA ligase utilizes ATP to make a phosophodiester bond between the 3′ hydroxyl group of one DNA strand and the 5′ phosphate group of another DNA strand.

Some of the advantages of blunt-end cloning are:

• simple design of the primers without any extra bases at the 5′ end of the primer
• does not require complementary sequence between insert and vector, so it is a universal cloning method
• versatile

Some of the limitations of blunt-end cloning are:

• non-directional cloning generates only 50% of inserts with the proper orientation
• both inserts and vectors do not have complementary 3′ or 5′ overhangs, resulting in little chance for association stability between insert and vector, leading to lower recombination efficiency when compared to sticky-end cloning
• prone to vector self-ligation
• can have more than one insert
• generating a clone with multiple inserts with the right orientation is low

When we did blunt-end cloning, we followed the protocol below:

We began by preparing the insert, which is any desired fragment of DNA to be cloned into a vector or plasmid. This was what the PCR step was for. Following this, we also prepared our primers (the same ones we used for the PCR). Then, we did phosphatase reaction of the vector prior to ligation. The PCR amplified fragments and restriction-digested fragments contain a 5′ terminal phosphate, which creates a significant problem in blunt-end ligation. Self-ligation increases the background with just the vector without the insert in it. However, this issue can be minimized by reducing the self-ligation by removing phosphates present at the 5′ terminus in the vector. Thus, phosphatase treatment will effectively reduce the background of empty clones by > 95%. Next, we did the ligation reaction, which is a simple process that was run overnight in the thermo-cycler.

Thursday

When blunt-end ligation was complete, we prepared competent cells for heat shock transformation, a fairly standard lab procedure. Clark got in a bit later today due to a doctor’s appointment, so I spent the start of the day washing and scanning my Western blot from yesterday with secondary antibody and TBST.

When Clark got here, he had already prepared competent cells of E. coli, specifically the DH5-Alpha strain. I have worked with this strain in previous years’ research, which proved helpful. Following this, we ran a standard heat-shock protocol in a 42 degrees water bath for 60 seconds. Following this, we put the cells in recovery. Finally, we streaked plates with the recovered cells and checked them the next day to see if the reaction had worked.

Friday

Our plates had a ton of colonies on them! I successfully edited a bacterial genome!

Today was a fairly simple day. I just cast 6 10% polyacrylamide gels using standard procedures I have described in previous blog posts.

Other than that, I worked on my poster and presented my slides from forever ago on a zoom meeting.

This marked the end of week 9!! I’m super excited for the symposium next week!