Methods 1 (lecture 3) DNA replication techniques Flashcards
cloning, subcloning, plasmids
To estimate the Tm of an oligonucleotide?
(#A + #T) x 2oC + (#G + #C) x 4oC = Tm of a primer
steps of bacterial plasmid DNA extraction?
- Grow bacteria overnight
2.Collect cells by centrifugation - Resuspend the cells in a buffer (contains glucose to maintain osmolarity, EDTA to chelate metal ions, de-stabilize membrane proteins, and inactivate DNases, and Tris to maintain pH)
- Lyse the cells with SDS (a detergent that makes holes in the membranes) plus NaOH (loosens cell walls, denatures the proteins, releases and denatures DNA)
- Neutralize the solution with potassium acetate that converts SDS to insoluble KDS: at neutral pH plasmid DNA re-anneals, whereas sheared chromosomal DNA remains single stranded and insoluble at high salt
- Centrifuge the sample to get rid of cellular debris, denatured proteins, KDS, and ssDNA chromosomal DNA
- The supernatant that contains plasmid DNA can then be either passed through a silica membrane (that will bind plasmid DNA under high salt conditions) or precipitated with alcohol and centrifuged to collect the DNA on the bottom of the tube
- Plasmid DNA (on a membrane or in a pellet) is washed with 70% ethanol
- Pure DNA (eluted from the membrane under low salt conditions or resuspended from a pellet) can then be used for downstream applications (PCR, restriction digest, etc.)
What is a multiple cloning site
Site in a plasmid contains recognition sequences for several different restriction enzymes
What is a restriction enzyme
enzymes which recognize a specific, usually palindromic, sequence (typically 4, 6 or rarely 8 bp long) in a plasmid
potential components of a bacterial plasmid
- multiple cloning site
- reporter for selection, can be interrupted by multiple cloning site
- origin of replication (controls copy number of plasmids)
- selectable marker/antibiotic resistance gene (select bacteria or other cells containing plasmid)
restriction enzymes bind ds or ss DNA?
dsDNA
what bonds do restriction enzymes cut?
phosphodiester
plasmid DNA becomes ___ after cutting?
linearized
if restriction enzymes cut plasmid in multiple places you generate ___
multiple dsDNA fragments
Restriction enzymes make 3 different types of cuts, what are they?
5’ overhangs, 3’ overhangs, blunt
what enzyme is used to re-attach blunt or sticky ends resulting from restriction enzyme cutting a plasmid?
DNA ligase
what are “sticky ends”?
staggered DNA ends with sequences complementary to one another
Would you use the same restriction enzyme or different to produce sticky ends with sequence similarity?
You could use one enzyme or two different enzyme to cut complimentary sites in two separate DNA molecules, the resulting sticky ends can be ligated by DNA ligase
What is subcloning?
cutting and ligating desired pieces of DNA together in a vector using a combination of restriction enzymes and DNA ligase
what is “competence” in regards to cloning?
competence is the ability of bacteria to accept foreign DNA (the ability to accept a plasmid)
two methods to make bacteria competent??
chemically- suspend in CaCl2 solution (permeabilize membrane) and subject cells to mild heat shock (make cells uptake DNA in a survival attempt) then allow recovery before antibiotic selection
electrically- wash to remove salts (ions will cause cell explosion essentially) and subject to short electric pulse
electroporation is more efficient than chemical competance
Why do bacteria maintain and not kick out plasmid?
plasmids impart antibiotic resistance (antitoxin to antibiotic)
reporter genes are often inserted where in a plasmid?
In the multiple cloning site
example of reporter genes used in plasmids?
LacZ code for b-galactosidase
b-galactosidase converts a substrate into a blue product
If a plasmid was inserted into a MCS containing LacZ would that colony be blue or white in the presence of the appropriate substrate?
If the plasmid was correctly inserted the substrate would stay white (as the LacZ gene would be interupted)
If plasmid was incorrectly inserted the colony would turn blue as you still have a functional LacZ gene
Describe the origin of replication of a plasmid (simply)
an AT-rich region of DNA where replication starts
If plasmid replication depends on host replication machinery is plasmid replication dependent or independent of host cell cycle?
dependent
If plasmid replication depends on plasmid-encoded replication factors (Rep proteins) is plasmid replication dependent or independent of host cell cycle?
independent
define replicon (in a plasmid)
origin of replication + all of its control elements
How do different origins or replication (ORIs) result in different copy numbers of plasmids?
Depending on how they are regulated you can have just a few to hundreds of copies of a plasmid (“relaxed” or “stringent” control) depending on whether the ori is positively regulated by RNA or proteins, respectively. A plasmid’s copy number has to do with the balance between positive and negative regulation and can be manipulated with mutations in the replicon (mutations in the control elements)
3 possible tests to confirm id of recombinant plasmid?
- restriction digest DNA from selected colony + gel electrophoresis
- PCR using gene specific primers
- sequence the insert and its junction with the vector
explain how restriction digest can be used to confirm id of recombinant plasmid?
- digest the plasmid with a restriction enzyme
- run the digested fragments on a gel
- you know the size of the correct fragment, there should be 2 bands of the correct size if sequence was inserted
How can one test the orientation of the insert in the plasmid by restriction analysis?
choose an enzyme/pair of enzymes to cut vector asymmetrically in the insert (cut the inside the insert + cut next to the insert in the vector), depending on the orientation of the insert you will get fragments of different sizes
explain how PCR can be used to confirm id of recombinant plasmid?
design primer that overlaps both the vector + the insert, the primer will only allow amplification is the insert is oriented correctly
(either forward vector primer and reverse insert specific primer or vice versa)
why is it important to orient insert correctly in plasmid?
Promoter must be upstream of insert to create the correct protein, or else you will make non-coding RNA (this noncoding RNA will be antisense and lead to RNAi mediated knockdown of gene of interest)
describe 4 subcloning scenarios (is regards to the ends of insert/vector cut)
- clone with 2 sticky ends
- clone with 2 different but compatible ends (use different restriction enzymes that give you compatible ends)
- clone with one sticky end and one blunt end (the ligation of the blunt end will be less efficient)
- clone with 2 blunt ends (compatible ends have inefficient ligation and directionality is cloning)
how to prevent self re-ligation of vector?
use alkaline phosphatase to de-phosphorylate the compatible vector ends, no p = no ligation. The insert will contain P on the 5’ ends so will be allowed to ligate, the 3’ end of the vector won’t ligate initially so there will be a nick (“nicked duplex”), the nick will be repaired by the bacteria
