Instructor: Ryan Schenck

Cell Biology Laboratory PCB3023L

Spring 2015

Lab 8
Screening clones
Introduction & Background:
Last week we ran our PCR products from our designed primers and if they
worked we continued by cloning the PCR products resulting from the DNA exraction
lab that were amplified with native Taq polymerase by ligating it into the cloning
plasmid pCRTMII-TOPO vector. This week we will be screening our clones to see if
our target DNA was successfully ligated and the transformation of the competent
E. coli cells worked properly.
This lab is going to be fairly straightforward, as we have already performed
PCR once and this time it isnt very different. If you recall from last week the vector
has a region that is designed to be used with M13F & M13R primers in order to
capture the PCR product that was inserted into the vector. These are the primers
we will be using to test the success of our cloning reactions. The process of
performing a PCR in order to screen our colonies that was grown overnight is
termed colony PCR (cPCR).
NOTES: You will need ice again. Obtain a cooler and fill with crushed ice.

Procedures:
1. We will be screening 7 colonies that were grown overnight + 1 negative
control.
2. Obtain your plates that were grown overnight.
3. Prepare the Master Mix (reagents shown below in the table) in a 0.5mL
centrifuge tube.
Prepare a 50L PCR reaction: (multiply out the volume per RXN and fill in the
Master Mix volumes, because we are screening 8 we will create a master mix with
enough reagents for two extra reactions to account for pipetting error; thus,
multiply the volum per RXN times 10 in order to get the appropriate volumes).
Reagent
Sterile H2O
50 mM MgCl

Volume per reaction (l)

Volume for Master Mix (l) (x8)

36.5
2

1.5

10X NH Buffer

10 M Primer F

2.5

10 M Primer R

2.5

Instructor: Ryan Schenck

10 mM dNTPs

Apex Taq Polymerase

Cell Biology Laboratory PCB3023L

Spring 2015

4. Aliquot out 50L of your master mix into each of the 8 wells.
5. Using the end of a STERILE toothpick pick the clones that are white. If you
have clones that are mostly white, use those; however, you will want the
white ones because this means that the lac Z gene was interrupted by the
insertion of, hopefully, our DNA.
a. Pick the clones by simply putting the end of the toothpick to the colony
and scraping it off.
6. Place the toothpick into one of your already prepared PCR wells. Move up
and down briefly into your reaction wells and then discard the toothpick.
Place the prepared samples into the freezer. Your instructor will run the
M13 PCR program in order to amplify your samples.