Control Strains and Plasmids

A plate with no colonies on it can mean four different things: the DNA was bad, the cells were not competent, the plates do not select, or something else went wrong. Without controls there is no way to tell which. The control stocks box in the −80 freezer holds what you need to find out.

When you need them

The carbenicillin plates stocked in the fridge are already proven. When you select with anything other than carbenicillin, the plates were poured for your experiment, so they are as unproven as your transformation. Set up all three controls beside your real plates.

The three controls

Each control answers a different question, so none of them can stand in for another.

Control	What you do	What it tells you
Restreak	Streak the control strain onto one of your plates	The plates select at all. A batch poured with dead antibiotic looks exactly like a failed transformation.
Positive	Transform the control plasmid into the same competent cells	The cells are competent and took up DNA.
Negative	The same competent cells, with no DNA added	The plate is not just growing untransformed cells.

The restreak is not the positive control. The positive tests the cells; the restreak tests the plates.

Getting them out of the freezer

The box is on the top shelf of the −80, a green box labelled control_stocks. Leave it in the freezer until you are ready, and don't hold the freezer door open while you look.

• Take out the plasmid tubes you need for your transformation.
• Take out a cell stock only when you are about to streak it, and put it straight back. It must not thaw.

Doing the restreak

1. Touch a pipette tip to the frozen surface of the cell stock. You only need a little, but you should be able to see cells on the tip.
2. Spread it across the plate.
3. Put the stock back in the box, and the box back in the −80.

The negative control plate gets competent cells with no DNA. Follow your lab sheet for which cells to use. In the video the negative plate is left empty; that is a mistake, corrected on screen.

What is in the box

Samples are named by the antibiotic they select on: A for carbenicillin/ampicillin, K for kanamycin, S for spectinomycin, E for erythromycin. The tube label is what is written on the tube itself.

Sample	Plasmid	Tube label	Selects on
A1	pJ01	pJ01-C 2°	carbenicillin / ampicillin
A2	pJ12	pJ12-A 2°	carbenicillin / ampicillin
A3	pJ19	pJ19-A 2°	carbenicillin / ampicillin
K1	p20N31	p20N31 2°	kanamycin
K2	pKQ-Upp	pKQ-Upp	kanamycin
S1	pTargetF	pTargetF	spectinomycin
E1	pTRKH3-slpGFP	pTRKH3-slpGFP	erythromycin

pJ01, pJ12 and pJ19 express GFP from promoters of different strengths: pJ12 is barely green, pJ01 medium, pJ19 bright.

Reading the plates

Restreak	Positive	Negative	What it means
grows	grows	no growth	Controls worked. Your own plates can be trusted.
no growth	—	—	Nothing could have grown on this batch of plates, so a blank plate from it tells you nothing.
grows	no growth	no growth	The cells are not competent.
grows	grows	grows	The plates are not killing untransformed cells.

Two more things to check:

• A lawn instead of colonies. If a transformation plate is covered edge to edge, nothing was selected: the cells were already resistant to that antibiotic. Mach1 is already resistant to erythromycin, so erythromycin experiments need a different strain. Use the one your lab sheet names.
• Colour under blue light. Colonies should look the way their plasmid predicts. The K1 control carries GFP and glows; S1 (pTargetF) has none and stays white. A plate of white colonies where you expected green ones means something else grew.