Question

1. What is the purpose of the PCR nucleotide mix added to your PCR reaction? 2. What is the purpose of Gel Green? What type of chemical is it? Molecularly how does it work? 3. Why are the restriction digest reactions mixed by flicking or pipetting, rather than vortexing? 4. In the ligation, hAPP can only insert in one orientation into the vector why is this? What is the role of hAPP in DNA replication? What is its role in the lab? How are they the same? 5. what is in the plate that allows for the screening, what different colors mean, and why? 6. how to read the band patterns on gel electrophoresis?

Answer 1

Ans 1

The PCR nucleotide mix is a premixed solution containing the sodium salts of four nucleotides namely dATP, dCTP, dGTP and dTTP.

PCR Nucleotide Mix has equimolar amounts of each deoxynucleotide triphosphates (dNTP) to ensure optimal PCR.

The dNTPs are critical for PCR efficacy as it is required to for DNA synthesis. PCR nucleotide mix added to the PCR provides all four dNTPs during PCR reaction.

Use of PCR nucleotide mix is advantageous over adding each dNTPs separately to the PCR reaction mixture as adding dNTPs as a mix simplifies pipetting steps and reduces the risk of contamination.

Ans 2

Gel Green is a green fluorescent nucleic acid dye used to stain either dsDNA, ssDNA or RNA in agarose gels. In molecular biology, visualizing nucleic acid on agarose gel is very common. Gel green is used with purpose to stain the nucleic acid that would enables visualizing of the nucleic acid bands on agarose gel

Gel green is a sensitive, stable and environmentally safe nucleic acid dye.

Like ethidium bromide, gel green is also an intercalating nucleic acid dye. Chemically, it is similar to acridinium orange dye as it has two acridinium orange subunits that are connected by a linear spacer.

As I mentioned, Gel green is an intercalating dye, it intercalates between the planner bases of DNA. In agarose gel wherever the nucleic acid would present, on staining the gel green will accumulate and give fluorescence that will helps in visualizing nucleic acid.

Ans 3

The restriction enzymes are protein. Mixing by vortexing would be a vigorous steps to protein that may hamper stability of the protein and would affect the functioning of restriction enzyme. Therefore, mixing of restriction digest mixture using vortex would be avoided. Gentle mixing by flipping or inverting the tubes containing restriction digestion mixture is recommended.

Suggestion

Agarose gel electrophoresis, the DNA molecules of low molecular weight migrates faster. Therefore, The DNA bands migrates less would be observed near the well on agarose gel and their molecular weight will be higher in comparison to the DNA band observed far from the well means migrates faster and their molecular weight is low.