Molecular Bio Flashcards
Plasmids
Double-stranded circular DNA molecules found in a cell, separate and apart from the chromosome of the cell
Many contain genes for antibiotic resistance
Plasmids ori
Origin of replication
Allows them to replicate inside the cell
If source DNA were not joined to a DNA molecule with an ori (vector), the source of DNA would not be replicated in cell
What makes a plasmid an attractive vector
If a plasmid can replicate to high copy number in a bacterial cell
PCR
Polymerase Chain Reaction
a way to replicate DNA in a test tube
Uses heat rather than helicase to separate DNA strands
DNA primers elimintae need for primase
One enzyme used, DNA polymerase from a thermophilic bacterium. Because it will not denature at high temps
Repeated cycle of strand separation, primer annealing, and new strand elongation can amplify a billion fold
Advantages of PCR
Ability to amplify tiny amounts of source DNA
Amplify a particular region of source DNA defined by the primers chosen
SPEEED
Restriction enzymes
Cut large pieces of DNA into smaller fragments.
Double-stranded DNA-specific endonucleases
Bind to certain sites on double helix and break phosphodiester bonds linking nucleotides
Also, make a staggered cut in DNA
Palindrome
Generally a 4-6 base sequence that a restriction enzyme recognizes and cuts
XbaI
Recognizes TCTAGA in DNA. When it cuts it leaves 4 base, single stranded ends
Cuts on the 3’ side of the T in both strands
resulting in
5’T CTAGA 3’
3’ AGATC T 5’
Sticky ends
Hydrogen bond to another identical sticky end by complementary base pairing
The two ends can then be covalently linked by DNA ligase
DNA ligase
If a plasmid is opened by cutting with a restriction enzyme, a DNA fragment from another organism cut with the same enzyme can be pasted into it. The foreign DNA will be replicated with the plasmid
Transformation
Only a few bacteria are naturally transformable. Most are not able to because cell membrane is impermeable to DNA
We use E. Coli as the recipient of our recombinant plasmit
pGEM-3Z
Plasmid that carries a gene for ampicillin resistance (bla), transformed cells can be selected by growth on medium containing ampicillin. Cells without it will be killed
Ampicillin
derivative of penicillin blocks synthesis of peptidoglycan, the main component of bacterial cell wall.
B-lactamase
bla produces this enzyme, which breaks down ampicillin before it can interfere with cell wall synthesis
MCS
Multiple cloning site
Present in pGEM-3Z
Region where a dozen different restriction enzymes have a unique cleavage site
MCS is near the start of the lacZ gene carried by the vector
Insertion of the foreign DNA in the MCS disrupts the lacZ gene and allows screening for reocmbinants by blue/white selection
Outcomes of transformation
Cell does not take up plasmid
Cell takes up plasmid without gfp gene
Cell takes up plasmid with gfp gene inserted
Cell dies on Amp plate because no bla gene
Cell grows on Amp plate but does not glow
Cell grows and glows
pGLO
Plasmid used as source for gfp, originally isolated from jellyfish
Modulates the green color of light
GFP
gfp regulated by presence of the sugar arabinose
Downstream of an araC-dependent promoter.
Will not be made unless arabinose is present
AraC protein
Made from a plasmid gene, serves as a repressor for GFP production in the absence of arabinose
When sugar is added to growth medium, AraC changes conformation and stimulates GFP synthesis
What was our aim
Take the gfp portion of pGLO and move it into pGEM-3Z
Use PCR to selectively amplify an 831 base pair fragment,
Trim that down using XbaI, and drop it into XbaI site of pGEM-3Z
This would separate gfp from the promoter that controls its expression (pBAD, where AraC protein binds
Making GFP expression independent of arabinose
Agarose gel electrophoresis
Helps predict the size of DNA fragments after being cut. Cast by melting agarose in buffer and pouring the warm liquid into a mold. Once the gel hardens, DNA samples can be loaded into wells at one end
An electric field is applied across the gel
DNA will migrate to the anode (positively charged electrode)
DNA must move through pores in the gel matrix
Short DNA fragments do this easier, so they migrate faster
Rate of migration is inversely proportional to the log of fragment length
Location of DNA in gel can be determined by staining gel with fluorescent dye that intercalate of the double helix
When UV light is shined on stained gel, DNA bands glow orange. The intensity of staining is proportional to the amount of DNA. Can tell concentration of DNA
Gel concentrations
Higher 2% agarose concentrations, lead to smaller pores for small fragments 100-1500 BP Lower concentrations (0.5%) best for large fragments (5,000-50,000)
Column Chromatography
Separates and purifies proteins based on some property they possess
Size, charge, hydrophobicity, or binding specificity
Cell extract loaded on a column containing bead matrix
Some proteins stick to the beads, others pass.
In a second pass, conditions can be changed, so some proteins no longer stick to the beads and elute.
GFP is hydrophobic
Under salt conditions, GFP will fold so the hydrophobic residues face inward and do not interact with water.
High salt- proteins will denature
Result is that GFP residues will be exposed
If passed over a column with hydrophobic beads, then it will stick to them strongly
As lower salt concentration, GFP will become normal again and no longer stick to beads
Column Chromatography Results
- Binding buffer: 2M (NH4)2SO4——GFP denatured. Sticks to beads
- Wash buffer 1: (NH4)2SO4—–GFP denatured. Sticks to beads
- Wash buffer 2: 0.4M (NH4)2SO4—-GFP denatured. Sticks to beads
- Elution buffer: 0M (NH4)2SO4—-GFP refolds. Washes off columns
Calculating Transformation Efficiency
To naturally transform E. Coli, mix with DNA, heat shock at 42 degrees C, small % will take in DNA
Our sample contains about 0.1 microgram of DNA
Part 1
Given solution of pGLO DNA. Some for transforming cells, others serve as template in PCR reaction to amplify gfp.
Premade PCR cocktail has enzyme, buffer, nucleotides, and primers
Part 1 Transformation
Add 2 microliters pGLO DNA to 200 microliters competent HB101 cells in microcentrifuge tube. Ice for 20 minutes
Transfer tubes to 42 degrees C water bath leave for 90 seconds. Put on ice for 90 seconds. Heat cold shock helps uptake of DNA
Transfer 100 microliter cells to nutrients plate with ampicillin
Spread remaining 100 microliter cells on plate with ampicillin and arabinose
Incubate at 37 degrees C for one day and kept at 4 degrees C
Part 1 PCR
Add 2 microliters pGLO DNA to 23 microliters premixed PCR cocktail in a sterile PCR tube.
Put tube in thermocycler. Run for 25 cycles
i) 1 minute at 95 degrees C to denature
ii) 1 minute at 52 degrees C to let primers anneal
iii) 1 minute at 72 degrees C for strand elongation
Stored in fridge
Part II PCR cleanup
Get ride of dNTPs, primers, etc may interfere with digestion
Add 125 microliters PBI buffer to PCR sample in PCR tube
Put combined 150 microliters into spin column in a 2 ml collection tube
Centrifuge for 1 minute at max speed. Linger fragments will remain bound to matrix while primers, nucleotides, etc will flow through
Discard liquid, keep tube
Wash column by adding 750 microliters PE buffer. Centrifuge for 1 min at max speed
Discard flow through and put column back
Centrifuge for 1 minute at max speed, ensuring dryness
Put column in clean 1.5 ml centrifuge tube. Discard collection tube
Add 30 microliters elution buffer to column and sit for 1 minute. Longer fragments will be released from matrix
Centrifuge for 1 min at max speed
PCR product is liquid in tube
Digestion
Dispense 16 microliters of cleaned PCR product into a microcentrifuge tube.
Add 4 microliters of XbaI enzyme plus buffer mix
Dispense 16 microliters pGEM-3Z vector into second tube
Add 4 microliters XbaI enzyme plus buffer mix
Incubate both at 37 degrees C for 45 minutes
Heat inactivation of XbaI
Both digestion tubes in 65 degrees C for 10 minutes
Ensures ligation is not undone
Ligation
Join fragment and vector Clean tube Transfer 8 microliter cut PCR product to tube Add 8 microliter cut vector DNA Add 4 microliter ligase and buffer mix Spin Incubate
Transformation 2
Add 200 microliters competent HB101 cells to ligation mix
Sit on ice 20+ minutes
Heat shock at 42 C for 1 minute
Transfer 100 microliters cells to Amp plate
Incubate
Purification of GFP
2 microcentrifuge tubes add 1 ml of pGLO to each
Spin at 10k for 5 minutes
Pour supernatant fluid in waste
Resuspend each pellet in 125 microliter TE plus lysozyme
Incubate at 37C for 15 minutes
Quick freeze cells in dry ice
Thaw in 37 C bath
SPin for 10 minutes
Combine supernatant fluid from both tubes
Add 250 binding buffer
Run whole volume through column and collect eluate in tube 1
Add 250 wash buffer 1 over column, collect in tube 2
Add 250 wash buffer 2, collect tube 3
Add 500 elution buffer, collect tube 4
Cap bottom, add 1ml equilibration buffer, cap top
Use UV light to see which has GFP
Assay for beta-lactamase
Transfer 100 microliters eluate from tubes to new tubes.
Put 100 microliters DI water in 5th tube
Add 100 microliters indicator solution to each tube/ Vortex
Check for clearing
