Colony PCR to confirm correct insert

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1. Obtain 3 PCR tubes per sample, and two extra for controls. Label the tubes on the top and sides as follows: one ‘u’ for the untransformed E. coli control, one “-” for the negative control with no E. coli cells. The rest you will label in the next step.

2. Choose three patches of transformed E. coli per sample that display good growth on the LB/kan plate. Record in your notebook which patches you have chosen from, and label the rest of the PCR tubes with the corresponding number and your sample ID.

3. Using a p20 pipette tip, transfer a small amount of each one of the patches into the bottom of the corresponding tube. Make sure the numbers match, i.e. the patch labeled 1 goes in the tube labeled 1.

4. Transfer some untransformed E. coli cells into the tube labeled ‘u’. These cells you can get from your non-selective control transformation plate that is located in the cold room.

5. Generate your PCR master mix for eight reactions (your five samples, two controls, plus one extra for loss of volume due to pipetting), using the following table: (see lab iPad)

6. Aliquot 25 µl of PCR master mix into each of the labeled PCR tubes. For tubes containing cells, use the pipette tip to gently stir the cells in with the PCR master mix.

7. Place labeled tubes in the PCR machine, run with the below protocol, and visualize on a 1% agarose gel. Think about why the initial denaturation step is longer here than in our previous PCR reaction.

Initial Denaturation 94C 10 min

Denature 95C 30 sec Anneal 51C 30 sec Elongate 72C calculate

Final Elongation 72C 1 min Hold 4C indefinite

You want to move forward with a sample with one clear, sharp band of the correct size (your insert size + 200 bp. Refer to the diagram below–your PCR will include all bases between the M13 forward and M13 reverse primers).

Start a 5mL overnight culture from the corresponding E. coli patch that can be used for plasmid extraction as well as creating a frozen E. coli stock (see below).