MCB4034 Mid-term

Why does there have to be a 1:2 ratio of plasmid to insert for the ligation reaction?

What we’re trying to do is balance competing reactions. The goal is to have one plasmid associating with one insert. If the ratio get upset, the plasmid might re-bond or a plasmid might insert itself where the insert was meant to go. If we have more inserts, the insert molecules might start sticking together.

However, we never see the plasmid-plasmid because when you have DNA molecules that are multiple plasmids stuck together, there are multiple origins of replication, and each origin of replication will work to replicate. Cells that have those structures inside are not able to replicate once they start to grow.

What are concatemers?

Concatemers form when inserts join together. These can potentially be inserted in the plasmid. (Which is what we were selecting for when looking at size.)

Slide 149 for week 3.

Are heterodimers better than homodimers? What’s the difference?

Should the extention step of PCR always be at 72C?

Primers

  • When you have a large difference between the two primers, your primer might bind to off target sequences.

  • The longer the primer, the more selective it is. (It’s a lot harder to find something like AACGTCG than just ATG, for example)

  • The max temp in PCR is 72, so a melting temp of 72 is kind of the max.

  • DNA polymerase needs 3’ group, template, and nucleotides (that’s why 3’ dimers are so much worse.)

Palindromes

The symmetry in the restriction enzymes is what make them recognizable.

PCR

MAKE SURE YOU KNOW PCR STEPS.

  • 2 min 94 (initial denaturation)

  • 1O sec at 94 (always fixed)

  • 20 sec at 56 (56 is variable depending on the melting temperature)

  • 45 sec at 72 (time is variable, depending on how much we want to extend. 1kb per 1 minute.)

  • 2 min 72 C (renaturation?)

  • Watch video from week 2

DNA purification

Elution is to get the DNA off of the silica membrane. Wash is intended to get off everything that we don’t want to carry forward (for example, salts, sugars, lipids.)

Sticky vs Blunt ends

Week 1

GFP (green fluorescent protein) originates from the jellyfish Aquorea victoria.

T. reesi is a fungus that produces cellulases used to breaks down biomass.

Week 2

Plasmids

  • Extrachromosomal circular DNA molecules

  • Usually 3-10 kb in size

  • Can be isolated from and introduced to bacteria

Isolation

  • Alkaline medium is used to lyse e. coli cells and denature the DNA, proteins, etc.

  • Acetate is added to renature the cell material, but most cell components will form a big clump and not properly renature.

  • Plasmid cells are small enough to renature to their original state.

  • This clumpy solution (egg drop soup!) gets centrifuged and the supernatant gets transferred to a tube with a spin filter. The plasmid DNA gets trapped on the silica membrane. The column is then washed with wash buffer to remove any contaminants. Finally, an elution buffer is added to release the plasmid DNA from the silica matrix. The final solution has the isolated plasmid DNA.

Restriction enzymes

  • Recognize specific sequences and “cut” like scissors.

  • To name restriction enzymes, the first letter is the genus (E for e coli), the next two letters are the species (co for coli) and then the strain designator and enzyme number (R1).

  • Restriction enzymes generate blunt and sticky ends.

    • Any two blunt ends can be joined, even if those DNA fragments were generated by different restriction enzymes.

    • Sticky ends are called sticky because they stick together via hydrogen bonds.

    • Sticky ends are also referred to as overhanging ends.

The LAC operon

  • Is only active under the presence of lactose

  • in the absence of lactose, repressors bind to the operator and prevents transcription.

  • When alolactose comes in contact with the repressor protein, it causes a comformational change that makes the repressor be unable to bind to the promoter. The polymerase is then able to use the lac operon to create the necessary enzymes to digest lactose.

Agarose gel electrophoresis

  • Dif. percentages are used to separate dif. sized fragments.

  • 1% is standard, and it separates (0.5-10 kb fragments)

  • 1.5-2% is used to separate 0.1 to 3kb fragments

  • 4% is the max concentration used and it separates 200bp fragments that differ in size by 10-20 bp.

PCR

Week 3

Ligase is the enzyme that forms phosphodiester bonds between two adjacent nucleotides.

Week 4

Week 5

Sanger sequencing

Mutations

Purines are Ashleighs and Grants (pure). Pyrimidines are C’s and T’s.

Mutations can be observed at the DNA level, or at the protein level.

At the DNA level:

  • deletion

  • insertion

  • substitution

    • transversion: changes purines to pyrimidines and vice versa (ex: A to T/ C to G)

    • transition: changes purines to purines and pyrimidines to pyrimidines (ex: A to G/ C to T)

At the protein level