What factors may have played a role in the separation of the dyes used in the gel electrophoresis?
Shape, size and degree of negative charge.
What is the recognition sequence for BamHI?
5'.....GGATCC.....3' or 3'....CCTAGG....5'
In a 5' to 3' direction, what sequence of bases represents the "sticky-ends for each"?
GATC for BamHI and AGCT for HindIII
pARA digestion will yield ___bps and will be ______ bps in length.
pKAN-R digestion will yield____fragments and will be_____bps and _____ bps
2, 4706, 702
Assume you were given a culture of bacteria carrying one or both of the plasmids. Design a simple experiment that you could use to determine wich of these plasmids pARA or pKAN-R in , the bacteria in the culture were carrying
You can put in enzymes to the bacteria; if it starts to glow, you will know it has the rfp.
Briefly define the term "Recombinant DNA"
A DNA molecule composed of ligated fragments taken for other DNA molecules.
What does the term ligation indicate?
Bonding it together
In order for two sticky ends to join together, what relationship needs to exist between them?
What is the importance of step one in this protocol?
Get rid of the enzymes so that they don't cut the DNA
helps bacterium make proteins encoded by genes inserted
Which antibiotic resistance gene is found in pARA?pKAN-R?
ampr is in pARA, kanr is in pKAN-R
One of the two original plasmids has been engineered to express a protein if the gene for that protein is inserted into a specific location. Which of the plasmids has been engineered for protein expression?
From what organism did the gene for mutant florescent protein come?
A sea anemone
Why do you suppose you are asked to set up two tubes without BamHI and HindIII?
They need enzyme control tubes to make sure the enzymes are doing their job
Why was it important to place the A+ and K+ tubes in the 70 degree water bath before setting up the ligation reaction?
The heat will deactivate any BamHI and HindIII restrictions that might be active
Could two rfp fragments join together and circularize in the ligase tube?
Describe how Hydrogen and Covalent bonds differ in strength and where, in the DNA molecule you could find them.
Hydrogen bonds are very weak and fail to hold together permanently. Covalent bonds are strong, formed by DNA ligase and complete the phosphodiester linkage. Hydrogen bonds form in the restriction enzymes; Covalent bonds make up the backbone of DNA
DNA ligase forms which bond?
During ligation, which of the bonds (covalent or Hydrogen form first?
Where do they form?
Which bonds form next and where do they form?
Hydrogen bonds form first in the restriction enzymes, covalent is next and forms the backbone of the DNA molecule.
How did your actual gel results compare to your gel predictions?
Some bands that were expected did not show up and some that were not expected did not show up.
Are there any bands, appearing in your gel photo that are unexpected?
Yes, my ligase looked like my marker because it had so many extra bands
WHAT COULD EXPLAIN THE ORIGIN OF THESE UNEXPECTED BANDS?
THere were multimers
Do you see evidence of the three plasmid forms in the uncut lanes?
YEs, they are in dfferent locations due to their size and shape
IS there evidence of more than one form of multimer?
Yes, there are more than one form of multimer. You can tell because they are in different locations.
Why are the ligated plasmids so close to the well?
Because they are larger and therefore unable to move as far as quickly
Is there evidence of a circularized 1404 bp fragment on the ligated lane?
There should have been but i did not see one because the substances sunk through the gel
Begin with the restriction digest of pARA and pKAN-R, briefly describe the steps required for construction your recombinant plasmid from pKAN-R and pARA restriction fragments. Explain your answer including the manipultions of these two plasmids annd their restriction fragments.
1. Use enzymes to cut plasmid
2. Insert the rfp gene from pKAN-R
3. Add bigger part from pARA to make pARA-R
4.Have it duplicate
WHat is "transformation"?
A process that places foreign DNA, like a plasmid, into a cell
What does it mean if cells are "competent"?
Cells are capable of taking up plasmid DNA
How will you know if a bacterium gets transformed with a plasmid containing the ampr gene?
it will be able to survive in an environment with ampicilin because it produces a chemical that will destroy ampicilin
What would be the size of a fragment carrying the ampr gene be?
How is the P+ culture treated differently form the P- culture?
Plasmid is not put into the P- culture
WHat is the purpose of the P- culture
to act as plasmid control
Predict growth if any on the plates from the experiment
LB plate: P-:yes P+:no
LB/ampplate:P- no; P+ yes
LB/amp/ara plate:P+ yes
What do all the cells growing on the LB/amp and LB/amp/ara plates have in common
What single restriction fragment must they all contain to grow on plates with ampicilin?
Would you expect that all the cells growing on the LB/amp/ara plate were transformed with the same plasmid? Explain.
No, not all the cells allow plasmids in through their plasma membranes
How might you determine which of the cells on the LB/amp/are plate contain pARA-R?
It will glow!
WHy did the red colonies appear on the LB/amp/ara plate and not the LB/amp plate?
Stops DNA loop from forming
WHY IS IT IMPORTANT THAT THE CELLS YOU COLLECT FROM YOUR MOUTH are mixed with chelex?
THe chelex beads will bind divalent magnesium (Mg++), which sere as cofactors for nucleases that will degrade the DNA sample. It allows you to seperate the genomic DNA from the cell debris
What is the purpose of boiling the cell sample?
To lyse the cells and liberate the DNA
What does PCR accomplish?
(Polymerase Chain Reaction)
TO produce many copies of a selected gene segment (locus) of DNA
THe primers that are used in this PCR are unique to what locus?
In tron region, tPA (tissue Plasminogen Activator), on chromosome 8
What is Alu element?
A 300 bp piece of DNA that has been randomly inserted throughout the genome. Called Alu because it carries a recognition sequence for Alu restriction enzyme
How many bp does Alu have?
Does everyone carry an Alu element in the region that we are amplifying by PCR? Explain.
No one knows because Because in evolution it is unnecessary to rid of the unused element
With respect to the tPA gene, how many genotypes are possible?
3: Alu+Alu+, Alu+Alu-, Alu-Alu-