Biochemistry summative assessment 1

insert, dna, interest, cloning vector, replicated, vector, host

the focus of molecular cloning is to … a … segment of … into a .. .. so that the dna segment can be … along with the … within a … organism

small carrier, dna, self replicated, plasmids, viruses, artifical

cloning vectors are … .. molecules made of … which are capable of … … and can be … , ……, and …. chromosomes

small, ori, replication, propagated, resistance, antibiotics, selectable marker, host, population, multiple cloning, restriction, insert

• plasmid

  • relatively ….

  • contain …. sequence

    • origin of ….

    • allows plasmid to be …. in host

  • carry genes that specify … to 1 or more …

    • … ….

      • which .. cells in a … of cells that carry the plasmid by using the marker

  • contain .. … sites where you have multiple …. enzymes sites where an … can be added

resuspend, buffer, p1, rnase a, degrade, contamination

Isolation of plasmid dna by a mini prep: Qiagen

1. … the cells in appropriate ….

   1. in buffer …. has … …. which … RNA inside cell , helps to avoid …..

lyse, denaturing, p2, sds, detergent, membrane, release, naoh, proteins, dna

Isolation of plasmid dna by a mini prep: Qiagen

2. … the cells using … conditions

   1. buffer …

   2. …. (sodium dodecyl sulfate) - ….. (denature proteins, disrupt cell …. which leads to … of cell contents

   3. …. - denatures … and ….

precipitate genomic, proteins, debris, membrane, n3, neutralizing, high salt, soluble, pellet

Isolation of plasmid dna by a mini prep: Qiagen

3. … …. DNA and denatured …., cell … and cell … components

   1. buffer …..

      1. … solution and provide .. … conditions

   2. plasmid DNA remains ….

      1. supernatant (solution above …)

centrifugation, supernatant

Isolation of plasmid dna by a mini prep: Qiagen

4. separate supernatant and precipitate

   1. …..

   2. plasmid in ….

purify, spin, silica, bind, high salt, spin, wash, low salt, pure

Isolation of plasmid dna by a mini prep: Qiagen

5. …. plasmid DNA from remaining cellular contents

   1. .. column → made of …

   2. plasmid DNA .. to spin column under .. .. conditions

   3. apply supernatant to … column

   4. … steps

   5. elute with .. … buffer

   6. … plasmid DNA → pET28

charged, electric field

electrophoresis - movement of … particles in an applied … …

separation, weights, size

agarose gel electrophoresis allows for the …. of DNA fragments based on their molecular …. (often referred to as their ….)

faster

smaller dna fragments run …. through the gel matrix than larger fragements

shape

the separation of dna in an agarose gel also depends on the … of the dna molecules as well

negative

what charge does dna have

known, linear

dna ladder - fragment of dna with … sizes (fragments are …. dna)

interculator, fluoresce

ethidium bromide - …..

…. under UV light

double, vivo, inside, compacting, faster, small

• supercoiled (SC)

  • … stranded DNA coiled (in …. - …. the cell)

    • … dna

  • moves ….. through gel than expected (….)

one, nicked, large, bulky, slower

• open circular (OC)

  • results when … strands gets ….

    • ….. and …

  • moves … in gel than expected

true, both

• linear

  • runs … to size

  • cut …. strands of plasmid by using RE

complementary, template, 18, 22, 50, 52, 58

Rules to consider on primer design:

• region of primer that is … to … ~ …-… nt (between primer and template)

• GC content ~ … % → melting point ~ ..-… °C

excise, insert, purify, ligate, ligase

Final steps of the cloning process:

• …. cut plasmid and … dna from gel

• … the dna fragments

• …. the fragments together using dna …