Lab 13: TA-Cloning of cDNA Fragments

What enzyme is used for the TOPO reaction?

Vacinnia virus topoisomerase enzyme inserts our DNA fragment into the TA cloning site of the plasmid vector utilizing the T-A overhang added by taq polymerase (A) and on the vector (T)

How much cDNA was added to the tube?

1.4µl was added

How much 6x high salt buffer was added and what were its contents?

0.35µl was added, and it had 1.2M NaCl and 0.06M MgCl2

How much of the TOPO plasmid vector was added?

10ng/µl, which was then incubated at room temperature for 30 minutes

After defrosting, how much bacteria on ice was added into a 1.5ml microfuge tube?

14µl was aliquoted and added

How much of the TOPO reaction containing the plasmid was added to the bacteria?

2.1µl

How long was the bacteria cells placed in a dry block heater for a heat shock and what was the temperature?

it was placed in the bry block heater for 30 seconds at 42°C and then placed back on ice

How much of the sterile SOC medium was added to the bacteria?

70µl

After adding the sterile SOC medium, why was it not placed back on ice?

To help recovery of the bacteria from the heat shock and to allow them to start making the antibiotic resistance gene product. There is no antibiotic in the SOC

Where was the tube placed after the SOC medium?

it was placed on the rotating wheel in the 37°C incubator for an hour.

How was a spreader made from a glass pipette?

It was heated into shape with a bunsen burner, and then placed into 70% ethanol for sterilization

Can a hot spreader be used for working with spreading the bacteria?

NO. It was cooled on the previously made LB agar plate, if used when it is too hot with bacteria, it will kill the bacteria

How was the bacteria spread?

Using the spreader from the glass pipette, it was spread gently on the top of the agar avoiding breaking the LB agar, this was done for a good minute to ensure it was as even as possible

Does the spreader have to be switched out in between bacteria or can the same one be used?

Since the sample is the same, the spreader does not need to be sterilized as long as it is not put down in between, if it is then re sterilization can be done by re heating it until it is not too hot and placed in 70% ethanol.

What was the incubation period for the plates and what was the temperature?

The plates were incubated overnight at 37°C, they were then removed and placed at room temperature to slow down the growth and then placed in the fridge.