Download Cloning homework_S11

You have finished Genetics and have decided to impress Josh with your molecular biology skills and
how much you learned in the class and lab so that you can work with him over the summer. You’ve decided that
cloning the drosophila gene, NOTCH, and probing for NOTCH expression in Drosophila embryos would be
just the thing to do. (Refer to your cloning handouts for information.)
While doing your background research you have found a few basic rules to consider for designing
primers for the PCR reaction:
1. Primers should be 17-28 bases in length.
2. They should have 50-60% GC content (so the total of G and C bases together should be 50-60% of
the total number of bases in your primer).
3. The melting temperature (Tm) should be between 55-80°C. (This is the temperature at which the
primer will denature. Your annealing temperature in the PCR reaction needs to be below this temperature by at
least 5°C and the two primers should not have extremely different Tm’s.)
4. Primers should end (3’) in a G or C, or CG or GC. (One or two C’s or G’s at the end promotes
stability during annealing. However, three or more C’s or G’s should be avoided should be avoided or you
could get ‘mispriming’ or annealing of your primer at G or C rich sequences.)
Using the ApE software on the computers in the lab (or a free download to your own), you will answer the
following questions; full sentences are not necessary. Be sure that any diagrams you include are labeled well
and include sufficient detail to clearly answer the question.
1. Design two primers that will amplify the majority of the largest exon of the NOTCH gene from w1118
genomic DNA. You have determined that the exons span nucleotides 1-1620, 1715-2024, 2097-2555, and 26232969 in the genomic DNA for a total of 2733 nucleotides in the cDNA sequence (the mRNA or coding
sequence for Notch). You have managed to find the cDNA sequence for NOTCH online and, as always, the
cDNA sequence given is the coding strand, written in the 5’ to 3’ direction.
1715
1
1620
2024
2097
2623
2969
2555
Genomic DNA
1
2733
cDNA
Figure 1. Schematic of NOTCH in genomic DNA compared to the cDNA sequence that was found and copied
from the NCBI website.
A) You need to have your primers made by a company, provide the sequences of the two primers you wish to
order (consider that DNA sequences are always written 5’ to 3’).
B) Highlight or circle the primer sequence on a printed text map (You can get this view by choosing text map
under the Enzyme drop down menu. The top strand in the text map is the coding sequence going 5’ to 3’).
C) What is the Tm and % GC for each primer? Write out a program for the thermocycler that you would use to
amplify the section of notch from the genomic DNA, clearly label all of the denaturation, annealing, and
extension steps. (Hint: Check the program we used the first week of the cloning project and consider what
changes might be needed.)
D) How large (in basepairs) is your expected PCR product? How can you determine if your PCR reaction
produced products other than what you expected? How will you determine the concentration of your PCR
product? Draw a labeled picture of your expected results.
2. Now that you have your PCR product, you want to clone it into pBluescript (pKS). Find the ‘unique’
restriction enzyme sites in pKS, those sites where the enzyme only cuts the plasmid DNA once.
A) Which RE enzyme site(s) would you try to engineer in your primers for amplifying the NOTCH gene? You
don’t have to actually worry about getting the restriction enzyme sequence into your primers you picked above
but be sure that the enzyme(s) that you choose will allow you to do Blue/white screening. (Hint, with the pKS
file open, go to Enzyme Selector and select all of the unique sites then choose graphic map or close. Then, go to
your window with the NOTCH gene and choose Enzyme Selector. Highlighted will be all of the enzymes that
cut pKS only once and you can read in () how many times those enzymes cut the insert.)
B) Will you be doing directional or non-directional cloning? What’s an advantage and disadvantage of each
type of cloning strategy?
3. Assume that you have put the appropriate restriction enzyme site(s) in your primers. If you have 2 restriction
enzyme sites that you will be using for your cloning just arbitrarily decide which primer will get which
restriction enzyme site. After cutting both the pKS and notch PCR product, you do a ligation followed by a
bacterial transformation into DH5α and plate your transformation mix onto LB amp IPTG X-gal media. You
end up with both blue and white colonies, nicely isolated on your plates the next morning.
A) Draw a diagram of the plasmids that you think the bacteria have in your transformation mix. Be sure to label
the ampicillin resistance gene, LacZ gene, your insert (if any) and its direction, and the restriction enzyme sites
used to digest your plasmid and PCR product in step 2. Include the specific nucleotides at which the restriction
enzymes cut. (Refer back to the pKS plasmid map provided in the handout “Cloning of genes form genomic
DNA”.)
B) Do you pick the blue or the white colonies to grow up and isolate the plasmid DNA for further testing?
Why?
C) How is selective pressure maintained in the overnight broth culture on the transformed bacterial cells grown
to isolate the plasmid DNA from (mini prep)? Why is it necessary to maintain this selective pressure?
4. Finally, you’ve finally grown up several bacterial colonies that you believe contains the recombinant plasmid,
pKS with the notch PCR product inserted. You isolate the plasmid DNA(pKS/notch) and want to check that the
plasmid really contains your insert and see which way the insert is orientated; for example, whether the 5’ end
of the notch PCR product is closer to the beginning of the LacZ gene or the T3 promoter.
A) Which of the following enzymes would you choose that would be easiest to confirm both, that notch is
inserted in the plasmid and it’s orientation in the plasmid – Cac81, DraIII, DrdI, or NcoI? (Hint: diagram both
pKS and your notch PCR fragment for each enzyme and the basepair # at which the enzyme cuts. Also include
the nucleotide(s) # where you expect the insert to be put into pKS - this was dependent on the enzyme or
enzymes you “placed” into your primers. Keeping the numbering systems for the vector and the insert
independent from each other can make the math easier in my opinion.)
B) Briefly, why will each of the other three enzymes not be as useful? Consider things like presence, size, and
orientation of the insert with respect to pKS.
C) Electrophorese the following samples on a 1% agarose gel containing the fluorescent DNA binding agent,
Gel Star (just like you have done in lab): 1 kb ladder (used in lab), undigested genomic DNA (as isolated from
the kit), your PCR product, pKS plasmid undigested, your isolated recombinant plasmid (pKS/notch)
undigested, pKS cut with the enzyme chosen in 4A, and your pKS/notch cut with the enzyme chose in 4A. Also
show the results of a digest if the notch PCR product were to get inserted in the opposite direction than
expected. Diagram what you expect to see on your gel for each sample, consider what differences there are
between undigested and digested plasmid DNA. Label your lanes (8) to indicate what the sample is and the
sizes of all of the bands.
Phew, do you think you made it into Josh’s lab?
****** Bonus Question: If you wanted to clone a portion of the first exon (>800 bp) using a naturally occurring
restriction enzyme(s) how would you do so? Be sure to include the specific enzyme(s) you would use. Would
you still be able to use the same enzyme as above (question 4) to check the orientation of the insert and if not
what other enzyme would work?
The link for the ApE program: http://www.biology.utah.edu/jorgensen/wayned/ape/ .
Here is a link for more detailed information on PCR primer design if you’re interested:
http://www.mcb.uct.ac.za/pcroptim.htm .