The end

Now I am done. I leave in a few hours for Botswana and South Africa - perfect timing. If I could find a source of isocitrate, I could do a functionality and specificity assay, but frankly I am satisfied with the protein on the gel. Yay! And I budgeted fairly well, using up most everything but some media and yeast genomic DNA preps. I did go over budget, spending $1100. Oh well. It was fun.

Thanks again to Mr. Rene Gillibert, Ms. Marietta Dunaway, Mr. Matthew Hamilton, Dr. Fang, Ms. Vicky Kim of Novagen, Ms. Janet Long of John Muir Regional Medical Center, Mr. Dunaway, the tech people at Epicentre, the sales people at Epicentre for offering a discount, and to the sales people at USB for offering a discount.

Purification and SDS-PAGE

I harvested after about 18 hours. Both cultures were clean and saturated. I used 5 mls of a culture in Sigma's His-Select iLap 5 ml purification column. The column is disposable and has only three steps: lyse, wash and elute.

I didn't order the required wash and elute buffers from Sigma because they were $150 and 100x more than I needed; so I made the buffers. Bad decision partly: my pH meter wasn't calibrating correctly, so I had to use a buffer calculator to figure the recipe with a fixed ionic strength. Something must have happend there, because the protein came out in the wash instead of the elute. At that, the corrosponding band in the cellular lysate was more intense than the "purified" band. But it was there, at the correct weight of 47kDa (actual is 46,562 Da).

Details on the SDS-PAGE: I ran the elution, a second elution (not suggested by Sigma), the wash flow-through, the lysed cell flow-through and the lysed cells (not the flow-trhough) on a 4-15% gradient Ready Gel from Bio-Rad courtesy of Ms. Marietta Dunaway. I used Novagen's Perfect Protein ladder 15-150 kDa. (The ladder is nice because it contains no oligosaccharides.)

Induction!

I innoculated two Novagen Overnight Express Auto Induction 125 ml cultures. As explained by Novagen,

The Overnight Express™ Autoinduction Systems enable regulated protein expression in E. coli without monitoring the culture or adding inducer during cell growth. These unique culture media are based on technology by F. William Studier at Brookhaven National Laboratory. ... Cell mass and target protein yield are often increased severalfold as compared with conventional protocols using induction with IPTG.

Hopefully my cultures are clean; I didn't use my clean air hood. I also skipped a staging step -- Novagen suggests starting with 30 mls and working up to 125 mls, but I'm trying to finish this by Saturday morning before I leave for a month for Africa.

Novagen recommends 16 hours of incubation at 37 before harvest. The flasks are on my orbital-hybrid microtitter shaker table at only 30 degrees; I plan to incubate overnight at this lower temperature. Novagen's product bulletin also says that the lower temperature may have other benefits. Cultures will have been incubating for 16 hours by 8:30 tomorrow; I'll harvest around noontime.

Transformation for Expression

Wednesday afternoon I transformed approx. 1 ng of a plasmid miniprep from colony culture #31 into Novagen's Tuner(DE3)pLacI. After a 60 minute outgrowth step (37 degrees with axial shaking - I don't have access to an orbital shaker presently), cells were spread on LB plates containing 1% glucose, chloramphenicol and carbenicillin and grown overnight.

Restriction Digestion

Gel shown are cultures 1, 2, 3 and 31 on a 1.3% TAE gel stained with Sybr Safe. Flanking lanes are Bio-Rad's 500 bp ladder (16 fragments from 500 to 8000 every 500 bp). Gel 2 is cultures 32, 33, 34 and 35, not shown.

I'm now working at home since school has ended. Over the past week I've done minipreps on eight cultures (grown in Teriffic Broth) and digested with Bgl II. Miniprep system is Sigma's GenElute Plasmid kit.

Calculated fragments
Correct at 557 and 4335
Reverse Orientation at 804 and 4088
No Insert at 3653

Clone #31 (fifth column shown at right) has about the correct size fragments, at approximately 4280 and 534. As far as I know, that is close enough to assume correct. My error probably comes from the fact that the bands were fairly wide on the gel image. Clones 2 and 3 have faint bands at 4280, and given that the heavy band is brighter, it might be safe to assume that the 557 bp band is present but too faint to see.

Two cultures had one band at about 2500 bp. I'm not sure what those bands were.

Ligation, Transformation, Update

PCR with 35 cycles was perfect. Details of run are in previous post.

I liagted the fragment yesterday (Wednesday) into the Novagen pETBlue-2 vector. I deactivated the Taq with chloroform and used 2 ul of the aqueous phase in the ligation. The fragment was blunted then ligated with T4 ligase. I transformed NovaBlue Singles (a K-12 derivative) and plated on LB with carbenicillin, tetracycline, X-gal and IPTG.

Today (Thursday) all the test insert cells were white and all test plasmid colonies were blue, as hoped for. I didn't have many colonies on the plate with the IDP2 plasmid. Nonetheless, the few I had were white. (I have more of the cells unplated [still in the tube that I transformed the cells in] stored at -86 C, in case I need to try replating the transformants. Each tube has 250 ul of SOC.) I picked six of the colonies and innoculated 3 ml TB cultures. The cultures are now in the incubator at 37 C with shaking at about 220 rpms.

Tomorrow, as long as the cultures are saturated, I will do minipreps on the six cultures, then do restriction digestions, then run on a gel.

Fingers crossed!

PCR

I finally got the desired product. Yields from 2 mM and 1.75 mM reactions were about equal. I used 50ng of genomic DNA in each and 0.5 uM of each primer, with a final volume of 50 ul. I tried one tube with 100ng of genomic DNA but didn't have a very good product. Program was hotstart 25 cycles of [1min at 94, 2min at 50, 3min at 72] with an initial denaturation and final extension. The bands were fairly faint, so I'm going to try increasing the program length to 40 cycles.

I'm going to take all my posts from the period when I was using a bad primer offline. If anyone desires to see them, contact me.

My mistake... Progress redux

Many thanks to Matt Hamilton and Jan-Feng Cheng at Lawrence Berkeley Labs. Matt set up a few new reactions for me with different enzymes and DNA prepped by their labs. Turns out, though... that I complimented the sequence for the reverse primer but forgot to reverse it! New primer is on its way.

Before this point, I'd been unable to get any products from my PCR reactions. I removed my detailed posts from this blog, though they are stored offline if anyone cares to see them.

Preparation of Yeast Genomic DNA

Epicentre MasterPure Yeast DNA Purification Kit.

Procedure overview:
1. Lyse yeast cells in the Yeast Cell Lysis Solution (non-enzymatic).
2. Precipitate and remove proteins using MPC Protein Precipitation Reagent.
3. Precipitate, wash, and resuspend the nucleic acids.

Used 1.3 ml A₆₀₀ = 12/ml. Yield was 816 ng/ul.