$547.68

biotin-16-dUTP re-ordered from Roche today...

Do we need to buy more lab equipment?

For the first time in a very long time, all four members of the Redfield lab were working at their benches at the same time yesterday. This meant more demand for equipment, in particular the benchtop microcentrifuge and the P-2 pipette...

In the past, we have discussed the possibility of ordering duplicates of these, but as demand cooled down, there was no need. This may happen again but I have been enquiring about pricing just in case.

Our microcentrifuge is from Eppendorf and is simple but great. The model we have has now been discontinued and an improved version has come out (model 5424):


This retails as USD 2660.

The Rainin CLASSIC PIPET 0.1-2UL would be USD 269.



Although we also have a P-10 pipette in the lab which covers the 0.5-10UL range so this can be used for most applications...

Still hunting for the biotinylated-dUTP...

Both Rosie and I have searched two -20 freezers but have had no luck... It seems odd that they have disappeared. The major complication is that we don't know what the box looks like or what was done with it since Rosie put it in the freezer two years ago!

A tube of 50 ul (50 nmol) costs $442 from Roche so we don't really want to have to order a new lot if we have some hiding somewhere...

50 nm beads...

... have been ordered from Miltenyi Biotec in California. I ordered the starter kit so that we get the beads and a magnet and stand.

Back to work!!

Today, Rosie, Josh and I had a brain-storming session about the preliminary data we would like to have before submitting our CHIR and NIH grant proposals.

One of the things that I'll be helping with will be useful for several of the planned experiments and has to do with binding DNA to beads. I had ordered streptavidin-coated magnetic beads for Rosie before I went on holiday and went to pick them up from Invitrogen this afternoon. The streptavidin on these beads will bind biotin, so if we attach biotin to DNA, we should be able to use the beads to pull down the DNA.

So I need to do three things:
1. Attach biotin to DNA
2. Bind the biotinylated DNA to the beads
3. Check whether H. influenzae cells bind this DNA and whether they can then be pulled down using the beads.

For step 1, I need to:
- locate the biotinylated nucleotides Rosie says we have
- find out the best way to attach them to my DNA (digest and fill in?)

For step 2, I need to:
- assess how well the biotinylated-DNA binds the beads
(quantify the amount of DNA bound to beads using a fluorescent dye - I must look into ordering some of this)
- I'll also check that non-biotinylated DNA doesn't bind the streptavidin-coated beads

For step 3, I need to:
- check how well the DNA on beads binds to H. influenzae cells (control: beads with no DNA on them. also compare competent and non-competent cells)

Strain collection

This morning I put all 76 ECOR strains into our strain collection database. I was surprised that this took me close to 1 hour! I have a few tasks to finish off today but then I'm off on holiday for a week in the Okanagan. Here is the weather forecast from enviro Canada, I can't wait!!!!!

Whole cell lysates

While I've got the ECOR strains growing in broth to make freezer stocks, I am also going to prepare whole cell lysates for all strains. I can then test these for PpdD production as soon as I get back from my holiday.

For background: Despite having all of the required genes, E. coli K-12 derivatives do not produce detectable levels of pilin (PpdD) or assemble pilus fibres on the cell surface . We have however shown that when we artificially turn on sxy, the cells make lots of pilin. I want to see if other E. coli strains already make at least some pilin, because this would mean that sxy is being expressed. In turn, this would be a good condition to test for natural competence.