Project for Biomolecular Principles: DNA
BIT 199A, Fall 2004
Subclone the upstream half of the SV40 promoter (the 209 bp EcoR1-HindIII fragment
from pCAT-P) into the multiple cloning site of pUC19.

The Insert is a fragment of DNA obtained from the plasmid pCAT-P.

pCAT-P is 4506 bp (base pairs)

The RE sites are given numbers to indicate their position relative to an
arbitrary bp #1

Eco RI sites in this plasmid are at 396 and 855

Hind III sites are at 605 and 2267

How many pieces should result from cutting the plasmid with both enzymes at
the same time?

How big are the fragments? (the sizes of DNA molecules are generally given
in numbers of base pairs, rather than in amu or Daltons, and so can be figured
by the difference between the position numbers – but be careful that you look
at the direction, because you have to take into consideration that position 4506
is right next to 1).

We will separate the fragments on an agarose gel, and cut out a block of
agarose containing the 209 bp fragment, and remove the agarose; that will be
our INSERT
The VECTOR that we are going to put the insert in is pUC19
Look at its map: the EcoRI and Hind III sites are at either end of the “polylinker cloning site
(396 – 454) on the pUC 19 map.
These sites are “unique” in pUC 19 (which means they each only occur once in the plasmid).
What happens when you cut pUC19 with EcoRI and Hind III?
The larger fragment has an EcoRI sticky end, and a Hind III sticky end (and most of the
original plasmid)
If we mix this with our 209 bp fragment (which also has one sticky end of each kind), it
should anneal in via the complementary overhanging bases.
If we then can add the enzyme ligase it will re-connect the sugar-phospate DNA backbone.
Before we do this ligation, however, it would be good to “clean up” the vector a bit:
1) remove the small 50 bp fragment (why?)
2) removing the two enzymes (why? )
Before we can set up a ligation, we need to quantitate the amount of insert and vector we have
(because we want to use appropriate relative amounts of insert and plasmid.)
Now we can set up the ligation, mixing insert, plasmid, ligase (and buffer) appropriately
Once we have completed the ligation, the plasmid can be replicated in a bacterial host.
We transform “competent” bacterial cells
Competent cells are ones that have been treated so that they take up DNA easily
Transformation is the process by which you get the plasmids into the bacteria
After transforming, we plate them out in selective media
Selective media is nutrient agar that contains ampicillin – the plasmid has a gene that
confers resistance to ampicillin. Only cells that have recived the plasmid have this
gene, and can grow. Each cell with plasmid grows up into a colony – all cells of that
colony have the same plasmid (and generally many copies of it ). Hence the gene is
“cloned”
Will all of the cells that grow contain the plasmid we are interested in (i.e., the one with the
209bp insert)? What are other possibilities?
Because other outcomes are possible, we need to test the plasmids directly, by looking at their
DNA. We can accomplish this a number of ways, and the method you choose often depends
on what you already know about the insert fragment.
First we need to grow up bacteria so that we get enough plasmid to work with.
We will purify the plasmid DNA away from the chromosomal DNA..
Now that we have a fair amount of DNA, we can digest with enzyme(s) that will help us
determine what each clone is. …. and so the analysis part begins ……
********************
An alternate cloning project, for more advanced students.
I would like to see if we can clone the gene encoding Green Fluorescent Protein into our
pUC19. Cloning it downstream from the LacZ promoter, and in the same direction, should
allow it to be expressed, and the bugs to glow. However, the company from whom I got the
plasmid containing the GFP did not make this easy, but I think we could do it like this:
Prepare insert: take pEGFP-N1 (or –N3) and cut with NotI or EagI
Blunt the end by filling in with Klenow + dNTP or by cutting with S1 exonuclease.
Heat 65 C 20 to kill the enzymes, extract with phenol:chloroform and EtOH ppt.
Resuspend in water. Digest with Hind III.
Run on gel. Cut out the piece that has the GFP coding sequence (look at the maps )
Prepare vector: (pUC19) cut with Sma I and HindIII (you will have to kill&/or clean-up these
enzymes). Also you will have to watch the temp b/c Sma I needs RT, Hind III needs 37 C.
Quantitate both, ligate and transform. (Check for functional GFP … do they glow?)
Pick plasmids, grow up in 3 mL batches, do mini-preps .