Instructor: Ryan SchenckCell Biology Laboratory PCB3023LSpring 2015Lab 8Screening 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). ReagentVolume per reaction (l)Volume for Master Mix (l) (x8)

Sterile H2O36.5

50 mM MgCl21.5

10X NH4 Buffer5

10 M Primer F2.5

10 M Primer R2.5

10 mM dNTPs1

Apex Taq Polymerase1

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.

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