L11 - DNA manipulation tools 2 Flashcards
(25 cards)
2 types of DNA digest analysis
recongition frequency;
analyse a sample via gel electrophoresis of DNA with a RE = see the frequency of different DNA lengths
Restriction mapping:
what does agarose gel elctrophereisis do
uses electrical charge to sepreate DNA mocleules on size and structure
produces bands
does low % agarose (0.5%) have large or small pores and what is this best to seperate
large pores
best for seperation of large fragments
what is ethidium bromide and what is its role in agarose gel elctrophereisis
stains DNA making it visible in UV light
= small molecule intercalates with DNA
wht stain do we use for gel elctrophereisis in our labs
Cybersafe blue
= stains it blue
non-mutagen stains are more commonly being used like thisbthat stain in colours instead of UV light
what kindve mathematical relationship links the movment of a DNA molecule in gel and its molcular mass
logathrmic
what do we use instead of the mathmaticaol relationship to work out the sizes of the DNA seen in gel elctrophereisis
DNA ladder
= provuides reference sizes and allows us to infer
estimate is good enough BUt if we wanted to be more accurate we could create a callibration curve
plot distance movced against size of DNA and then add in our results for accurate size
when cutting a plasmid and running it on a gel what are the 3 comnformations you can view ?
Nicked/open circle:
only 1 strand is cut of the plasmid = lowest mobility
Linear:
both strands are cut
Supercoiled/superhelical form = neither strand is cut and still in native form = highest mobility
what is agarose gel elctrophereisis
complex network of pores whiuch DNA must travel through towards positove charge
smaller the molcule the fatser and easier it nmoves through pores
= circular and nicked DNA does NOT move according to their size
what is SDS-PAGE
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
similar to agarose forms a network of pores for proteins to move through based on size
SDS is a denaturant added to turn protein molecule into an unstructured linear chain
= allows gel to seperate based only on length
the gel is syained to allow visualisation
native and denaturing forms of gel electrophoresis
denaturing:
analysids of molecules in primary structure/linear
seperates only on length and mass/charge ratio
= SDS for proteins or Urea for DNA
Native:
analysis of the natural stuctures of the molcules
the physical size of the assembled/foled molecules affects the mobility
= downside of this is its difficult to predict
what is T4 DNA ligase
purified from infected E.coli
can repair nicks BUT more importantly can repair blunt or sticky ends if base pairings are compatabible
requires 5’ phosphate 3’ hydroxyl and ATP
describe the 3 steps of T4 ligase forming a phosphodiester bond in nicked DNA
- Ligase is slef-adenylated by reacting with ATP
- Adenyl group transfers onto 5’ phosphate
- phosphodiester bonds forms between the 5’ adenylated donot and the 3’ hydorxyl acceptor
why are sticky ends easier to ligate together
hydrogen bonds between the complimnetarty overhanging strands hols the molecules in place while ligation takes place
how could we shorten DNA ends and why would we do this
‘chewing back’ 3’ overhangs
DNA polymerase possess 3’ –> 5’ exonuclease activity
= removes nucleotides from 3’ end
= only works on ss DNA and will stop once the overhang is chewed off
this can be done to remove overahangs to allow ligation with a blunt ended vector
how could we lengthen DNA ends and why would we do this
‘filling in’ overhangs to produce blunt ends
polymerases possess 5’—> 3’ polymerase activity
= adds nucletides to 3’ end
this can be done to remove overahangs to allow ligation with a blunt ended vector
whyb is Tdt polymerase special
Template independant polymerase
= can add dNTPs/deoxynucleotides without a template strand
why would we use the template independant TdT polymerase
adds on dNTPs to 3’ hydroxyl ends
for:
create compataible sticky ends for ligation
creation of ‘homopolymer’ compatible tails
= add on the same nucletide continously to one end and the opposite for the other DNA strand
you get 2 compatible tails TTTTT and AAAAA fro exmaple for ligation
ORR labvel ends with flourescent /radioactove nucletides
what is alkaline phosphatase used for
removes phosphates from 5’ end of DNA and RNA
= dephosphorylated ends prevents self-religation of a cut piece of DNA/plasmid
3’ OH group cannot from bond
what is T4 polybnucleotide kinase and what is it used for
phosphorylates 5’ ends of DNA and RNA
= allows ligation and formation of phosphodiester bonds
= can label 5’ ends with radioactive or flourescent ends
why might there be additional bands in my restriction digest
contamination with another enzyme/nuclease
‘Star’ activity = enzyme binding to off-target site
why might a restriction digest run as a smear on gel
DNA is shifting due to the restiction enzyme still being bound to it
why might my restriction digest be incomplete
enzyme is dead or too little added
incorrect buffer or not fully thawed from being on ice
why might there be only a few or no colonies in gene cloning practical
correct ligation:
ligase is dead
incorrect buffer
ineffectrient phosphorylation:
no ATP
= if colonies are not surving its because they do not have the gene for antimicorbrial reistamnce or whatever selection method is being used
