Molecular Techniques I: DNA Flashcards Preview

Cell and Molecular Biology > Molecular Techniques I: DNA > Flashcards

Flashcards in Molecular Techniques I: DNA Deck (27)
Loading flashcards...
1

Types of dna techniques

"to clone Paul C.Rxn from down South, go to the Library and return with the Sequence"

Cloning

PCR

Southern Blot

DNA library

Sequencing

2

Requirements for cloning

Plasmid

Eukaryotic DNA

Restriction enzymes (endonuclease)

Ligase

 

3

Cloning procedure

4

Plasmid requirements

Origin of replication

Selectable marker

Restriction site

5

Gimme: what do you use for foreign dna that's more than 20kb?

Bacterial Artificial Chromosome 

Yeast Artificial Chromosome

6

Clustered restriction sites are called

Multi-cloning regions

7

How do restriction enzymes work?

Cut at 4-6bp sequences (aka restriction sites) that are palindromic

8

Possible ligation products after cloning

9

Transformation requirements

Competent cells (electrocompetent or heat-shock based)

Electroporation/Heat shock

Antibiotic selection

Colony screening

10

Transformation procedure

11

Colony screening types

Blue white screening

Hybridization

(Colony PCR w/ flanking primers and gel)

12

Blue white screening procedure

13

Hybridization procedure for colony screening

14

How do you make a DNA/RNA probe?

Find 6-7 aas in a row in a region with the least degeneracy

Total probe size = 18-21 nucleotides long

Radiolabel 5' end

15

Polymerase chain reaction procedure

16

Calculation of copy number after PCR

2^n where n = # ds strands generated/cycle number +1

17

Describe how you would use PCR to identify mutations in known genes using the CFTR gene as an example

(check sheet for answer)

Need to design primers that flank the normal region of the gene and the deleted/mutated region of the gene, amplify DNA then run a gel

If HM normal, gel will show ONE large band

if HT, gel will show 2 bands (one large, one small)

if HM mutant, gel will show ONE small band (mutated region)

18

Southern Blotting procedure

19

SNP-based Restriction fragment length polymorphism analysis

SNP abolishes restriction enzyme site

 

20

STR based RLFP analysis concept

21

DNA library types

cDNA library construction process

Genomic and cdna

 

22

Sanger sequencing (old school) concept and procedure

Based on chain termination (via ddNTPS) and 4 separate reaction mixtures

23

Sanger sequencing (new school) procedure

(still chain termination) One reaction and capillary gel. Signal read by laser since ddNTPs have flourophore

24

Next Generation sequencing procedure

Why is it called "massively parallel?"

25

Ion torrent Next Gen Platform

26

Reverse termination/Bridge PCR (Illumina) NExt Gen

27

Bridge PCR for Illumina