skills USA Biotechnology

where does agarose come from?

seaweed

what type of substance is agarose?

carbohydrate

what temperature is it safe to pour agarose?

55 - 60 ^oC

why does DNA move during electrophoresis?

it has a negative phosphate groups

which runs faster (farther in the gel) smaller or larger pieces?

smaller pieces

which direction does DNA move?

run to red! red is the anode (positive) side

where should your wells sit?

the black side. the black side is the cathode (negative)

what is something that affects how something migrates?

size (mass), shape, and charge

what is the purpose for the buffer?

to conduct electricity (it has ions) and maintain pH

where do the bubbles come from?

electrolysis of water (splitting water into hydrogen and oxygen gas, hydrogen is at the black end oxygen at the red)

why are DNA samples in a blue loading buffer?

so we can see the sample and it has a high density so it weighs down the sample so it sinks in the well

what is a big disadvantage of DNA stains?

they bind DNA so they bind your DNA and are carcinogenic

what would happen if you made your gel with water instead of buffer?

it would get really hot and possibly melt because it would be resistant to the electricity instead of conducting

facts about enzymes

• naturally occurring, found in bacteria, different types of bacteria have different restriction enzymes, they are names after bacteria they come from

• discovered in bacteria in 1962, restriction enzymes are made to protect bacteria from foreign DNA, have a method of marking their own DNAas being “self”, any DNA not recognized as self is digested into smaller pieces by the restriction enzymes

• restriction enzymes search for exact sequences of a defined length, some enzymes recognize sequences 4bp long, some 6, and still others 8 or more, one of the common features of most enzyme recognition sites is that they are pelindromes, a palindrome is a sequence which is read the same on both strands in the 5’ —> 3’ direction

• also called restriction endonucleases

what is star activity?

when restriction enzymes cut non-specifically (not just at their sites)

when can star activity happen?

• too much enzyme

• too high of a pH

• low salt conditions

• too much glycerol or organic solvents like DMSO

• incorrect ions present like Mg⁺² or Cu⁺² or Zn⁺²

information about a plasmid

• plasmids are small, double stranded circular pieces of DNA that exist like an extra chrmomosome

• found in bacteria naturally

• contain the following parts

  • origin of replication - so they can be copied

  • antibiotic resistance gene - so bacteria that contain them can be selected for

  • MCS (multi cloning site) - so you can put genes into the plasmid

  • promoter region - to make the product of your gene