Restriction map of vector multiple cloning site:
T7 -> -Apa I - Aat II - Sph I - Xba I - Xho I - EcoRI - Kpn I - Sma I - Csp45 I - Cla I - Hind III - BamHI - Sac I - BstX I - Nsi I - <- SP6
1. You have a fragment of DNA that has been cut with Sph I and Not I. Describe how you would clone it into the multiple cloning site shown above.
2. You need to clone a fragment of DNA that is flanked by the Hind III and Pst I sites. Describe how you would cut & clone it into the multiple cloning site shown above.
3. In the creation of a genomic library, you have isolated genomic DNA and partially cleaved it with the restriction enzyme Sau3A I which has a four base recognition site. The time-limited cleavage generally gives a random set of fragments that can be size selected to give clones with inserts around 1000 bp. Given the vector above, how would you clone these fragments?
4. You have a diverse number of enzymes in your freezer but find that you are
out of Csp45 I. You have isolated fragments cut with Csp45I and need to cut
the vector above at the Csp45 I site in order to finish cloning these fragments
into the vector. There are at least two simple ways to accomplish this. Please
describe one of them.
5. You need to do a quick subcloning experiment that will transfer the insert
from the vector shown below into the vector shown above. You do not want to
gel purify any fragments but just cut each plasmid, mix them together, ligate,
transform and select for the proper recombinants. Select the best enzymes to
cut the vector above with and the plasmid below with to ensure that only the
recombinant vector above will survive but the plasmid below will not. The insert
contains restriction sites for Pvu II, EcoRI and Cla I.
Plasmid - Xho I - Pvu II - Hind III - Insert - Hind III - EcoRI - Cla I - Plasmid