mPCR result discussion

Ray came to our lab and ask for some sheep blood, and also asked if the mPCR result is right as being expected. As the result shows that the swabs MedLab gave me were NOT pure-cultured and there were several different species in one swab, I wondered why Ray told me I didn't need to isolate another pure culture. However, Ray said those non-pure-cultured swabs were "understandable"?! What the earth does he mean??

Holiday

Today is Auckland anniversary (nothing to do with hamilton though) and is off.

Cost cut in a lab

Equipments and chemicals in a lab are normally expensive. Actually one can't find any exception on earth. Thus lab-workers may have to figure out their own way to cut cost as much as possible. I think re-using tips and buffers may be one of the most common manners in each lab. Here I find some interesting examples of know-how (from The Scientist, Volume20, Issue 1, Page 55): 1. Stay away from the latest technical developments, and make do with the previous generation of equipment. 2. Make your own reagents. # This is why I make my own TBE(10x), TE(10x) buffers instead of purchasing new ones for my pcr. I see some people even make their own Taq and markers as well!! (amazing! How do they do that?!) 3. Grow your own cells. 4. Ask for free samples. # Like the GoTaq from Promega...but actually it's been too late; Kerry'd ordered Eppendorf HotMasterMix before the in vitro sales came to us. Anyway, can GoTaq perform hotstart? 5. Buy fusion antibodies. 6. Avoid top-shelf reagents. # So I don't need Taq from Qiagen. Also we may extract nucleic acid with traditional method (phenol/chloroform method) which is much less expensive than DNA/RNA prep kits (e.g. spin column). 7. Scale down recipes. # We usually do this one. 8. Reuse antibodies. # I did this when I was performing Western blotting in ex-lab. 9. Avoid chamber slides. 10. Autoclave, don't filter. # ...but filter sterilization is still needed when autoclave is unsuitable (e.g. enzymes). 11. Split the use and the cost of research animals. 12. Reuse, reuse, reuse. # Hah! This is gold-standard method for budget saving!! Reuse agarose gel, electrophoresis buffer and tips, etc. 13. Buy plastic bagged instead of racked. # Can anybody suggest where we can purchase plastic BAGGED filter tips?? 14. Use sterile items only for sterile techniques. 15. Get biotech company leftovers. 16. Create the equipment you want. # In other words, "order-made".

SB buffer

Last time when I wanna make agarose gel and run electrophoresis, Ray didn't use TAE or TBE buffer but SB buffer. He said TAE/TBE is expensive. After that I did some brief search about SB buffer and find something below: 1. Tris is the expensive part of TAE/TBE and on the other hand SB is relatively cheaper than those buffers contain Tris. 2. With SB the electrophoresis time can be cut down to 1/3 with high voltage (275V, which is 3-5x of Tris gels!) yet low heat generation than ordinary Tris-based gels. I see some guys run gels at 200V for 0.5hr with the same resolution and separation as 2 hours at 50V in TBE. 3. DNA bands with SB would be sharper than with Tris-based gels.

Tris gel (110V)	SB gel (275V)

4. SB wouldn't salt-out like high concentration TBE normally does. 5. The recipe of SB: NaOH (10mM) pH adjusted to 8.5 with boric acid (works out at ~2.6g/L). That's cheap! # SB is first reported in: Brody JR, Kern SE.(2004).Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.36(2):214-6. Erratum in: Biotechniques. 2005 Jan;38(1):60

New host visiting

Just come back from new host visiting now. It is Jason's host at the moment - may until February. Basically it's a small (at least to my mind it's smaller than the Turners) yet lovely house. The host is a fairly talkative American lady and her English is quite easy to understand. I find her a bit naughty as well. :P By the way, to move to this new host or not has nothing to do with my parents and Tzuchi foundation's "pursuation". This is MY DECISION.

Fake stem cells 2

Hwang Woo-suk's news on CNN: Cloning scientist: Forgive me Cloning scientist asks forgiveness Stem cell fakery called 'criminal act in academia' School sorry over stem cell fraud School: Human stem cell work faked School: Human stem cell work faked In S. Korea, 'no' gets failing grade Panel: No data for stem cell claim Panel says stem cell work faked Panel says stem cell work faked Stem cell expert: Work is not fake 'Long road' for stem cell research

Fake stem cells

The following news about Hwang Woo-suk are all from BBC News: Korea cloning expert questioned S Korea probes stem cell trials Korea cloning researchers quizzed Journal retracts Hwang research Hwang apologises to South Koreans S Korea cloning research was fake Korea's national shock at scandal Science takes stock after clone row The fall of a scientific 'rock star' Science will stick with peer review South Korea's cloning controversy

I have a dream

I usually make strange dreams at night. I didn't plan about content of dream before sleep, but somehow the contents usually have something to do with my real life in some ways. These are some of them: 1. Doing resesarch work in my ex-lab (forgot its content) 2. I was on a fisherman's boat with its owner (he is one of the fishermans work at our harbour when I was serving as a CGA member. I've forgotten his name though). I wasn't a CGA member in that dream but just sitting on the boat and the fisherman was sailing his boat on a river - like Yellow River in China. As I was sitting at the edge of the boat, the edge crashed suddenly and needless to say I fell down into the water as well. Fortunately the water was not deep. 3. This one could be one of strangest dreams. It was in a barrak somewhere and there were a row of soldiers standing in front of me - their chief. I saw I was blaming at one of them. I couldn't see his face but I can still remember his voice. I: ~!@#$%^&*(!!! He: You can't talk me like this! @#$%^&*... I: Fxxx!! I've been standing you for long time but you still ~!@#$%^&*(^$...!!! I saw this dream just after I entered CGA but hadn't met those guys standing in front of me and didn't concern about it too much; and amazingly they eventually were in my barrack months later. When one of the guys came to my barrack, I had a feeling this was not the first time to see him but actually we never see each other before and I couldn't figure out why I had that feeling that time. ...and one day the situation came to true - almost the same. I was almost going to blame at that guy, and at the same time this dream came up to my mind and realized - it's him!! No wonder I felt I'd seen him before we exactly meet each other!! (Wow! I'm a fortune teller!!) 4. I've forgotten the detail of another one, but I can still remember Peter (my honoured boss) was also in that dream as well!! ...and last night Yuki showed up in my dream. I was gonna leave somewhere (?! a bit similar to my real situation) and somehow he knew one of my secret - a true secret in the real world that I've been keeping from him! Dream is really amazing. Anybody can explain these phenomena?

Metabolic pathways and others

Find a useful (yet quite large-sized) biochemical Metabolic Pathways Map on Sigma's website accidently. There're also some useful tutoring animations of biochemical reactions (e.g. TCA cycle, glycolysis, etc). PS: How to print them?? ^.^;;

Re-make oligo

Kerry showed me an email from Sigma which says some of their recent oligos may not meet their quality control and, unfortunately, one of my oligo primers is concerned as well (primer CFF) and asking me if I'd like them to re-make the issued oligo. Have a look on my mPCR results yesterday, I can't see any problem with the C. fetus band. Regardless of this, I still decide to re-make it (it should pay nothing, after all) :P The new oligo is supposed to arrive Wednesday next week.

Multiplex PCR

I've done PCR before (Er...I mean 4-5 years ago), but this is my first time to do multiplex one. Actually their principles are quite similar - except the number of primer pairs). The result shows that of 14 Campylobacter isolates (plus 1 negative control), most of them are C. jejuni which is followed by C. fetus and then C. coli; no other species (such as C. upsaliensis and C. lari). # It looks like the samples from Medlab are not necessarily pure cultures... :( # Method n Material: DNA extraction: boiling method with chloroform purification Taq: Eppendorf HotMaster Mix Thermocycler: MJR PTC-100 Electrophoresis system: ????? Electrophoresis gel: agarose dissolve in SB buffer (with Ethidium Bromide dissolved) Electrophoresis buffer: SB buffer (not TAE or TBE buffer!?) Gel imaging system: ??????

Centrifuge and absorbance ratio

Ethanol precipitation with Ray's centrifuge today. I reckon Ray's protocols are somewhat strange than normal ones. Yesterday I also had a feeling he might not check nucleotide purity (absorbance ratio test) after DNA extraction. ...and I'm right - they don't. "That's textbook!" said Ray. ...I can see what he means, but at the same time I still feel odd without doing QC...

DNA extraction

To do PCR, DNA extraction is unavoidable, and thus I have to extract that of Campylobacter isolates. As I'm not using these DNA for SEQUENCING or CLONING but just differentiate what species they are, I wouldn't spend too much time on extraction and purification, hence I choose one of the simplest ways - boiling method - to do my work today. (Lysis method requires some more chemicals and that would increase my expense) 1. Boil Campys Normally I have to boil bacteria 10 min and suddenly chill on ice for another 10 min, but unfortunately the lids of eppendorf tubes pop out as the air heated up and I tried hardly to tie them up with celotapes (don't work) and sterilize tapes (more or less work) 2. Chloroform purification Unfortunately our chloroform has run out at the moment and Kerry borrowed a small bottle of chloroform (~80mL) from another lab. As this is a corrosive material, I'll have to record how much I use and write down on a track sheet, and collect the waste into another empty 2.5L winchester bottle. Eventually I used 2.8mL. Only 2.8mL chloroform in a big 2.5L winchester bottle... Looks stupid... -.-;; 3. Add NaOAc and Ethanol precipitate DNA Here's really a problem. I've got 1mL of ethanol and DNA sample in each eppendorf tube and have to centrifuge 5min - with the horizontal centrifuge in our lab. Oddly, the lid pop out and my samples almost flow out! Concerning my sample, I asked Kerry to change the roter into a "normal" one and, for in case, I tried to centrifuge some tubes with water to see if it's there's any problem. But alas! After testing 5 min centrifuge with water, the water gets warm and the roter is shockingly hot - actually it's untouchable! What would happen if ethanol is heated in capped tubes? (you can imagine how dangerous it is). This centrifuge is said to be around 15 years old... As this is unusable, I'd have to go down to Ray's lab and centrifuge my samples there... 4. Ethanol precipitation not needed?! In Ray's place, he told me I just need to use chloroform and then run pcr - ethanol not needed! This really surprised shocked me. Now I see his protocols are somehow different from normal ones...

Sales

I'm not handling genetics things too much in my research work, but a presentative from a chemical company still comes to see me and have a short meeting. Vanessa - the female PR - is from in vitro company and asked me if I could tell her what my work is about. We also talk about PCR and, somehow, Promega. She said she'd send me a catalogue of Promega for the year 2006 and GoTaq as a sample. She then went to Raywen's lab after that.

Andrew quits job again

As we'd suspected, the pay is exactly too low. Andrew said the job is too stiff and he wouldn't do it again and, as usual, starts complaining about his job. It is definitely better than equivalent to nothing. I wouldn't be too surprise to see Andrew changes his mind within only one day work; however I'm quite surprised when everyone feels the pay is too low whereas he still says it's better than nothing! :(

MIC with McFarland standard

Quite surprisingly, this time the result is quite consistant. Although the MICs are still slightly higher than with agar dilution method, it's now within an acceptable range. And also, this modified method has saved me quite a lots of effort and thus I may speed up my work on campys (decrease my working time from 4-5 days to only 2-3 days for each test!) Cool!!

Andrew's new job - picking onions ?!

Andrew has found a new job last Friday, and today is his first day to go. Needless to say, it's hard. Workers are supposed to pick onions under a beating sunshine and it is said to be paid by meters (not by hours?!) Andrew has went 77 meters today and when he came back home, he's almost been covered by dust (and so is our bathroom...) Andrew guess the pay might be 35 cents per meter, and of course all of us are surprised of its low pay. 35 cents!! Is this a joke?! Whereas he says it's better than nothing, but I'd say it's equivalent to nothing!! We asked him to ask his company how much the pay exactly is tomorrow.

McFarland standard <-> Optical Density

I've been having some difficulties on determining inoculum density as there's no golden standard for Campy. As the inioculu density may vary the final result of MIC significantly, we are supposed to control it consistently (and reasonably). Normally we can do "Total Count" to evaluate the total number of our bacteria in absorbance adjusted (0.5 absorbance at 540nm wavelength) and dilute it to particular concentration which is followed by loading 5-10 ul of it (triplex) on agar plate to see how many CFU there are. Here are two problems with my Campys. First, some of my Campys migrate on agar plates. This really make total count hard to achieve as several colonies might combine each other and it's impossible to count them. To overcome this I dilute the inoculum several more times so that there wouldn't be too many colonies on each observing unit (thus reduce the chance of merge). Second, and also the most annoying one, as Campys grow much slower than other cultures, I got to incubate quite a long time (2-3 days) to make the cell quantity achieves 0.5 absorbance. That really takes too long!! So, this time I modify my method: 1. Adjust inoculum concentration to 0.5 McFarland standard (~ 0.08 absorbance at 625nm wavelength) 2. Consider the antibacterial activity might reduce faster within aquatic condition (broth dilution method) than in semisolid condition (agar dilution), the incubation time is reduced to 1 day.

Before IELTS

Jason is gonna sit IELTS test tomorrow and gets me a phone call tonight. Apparently he's nervous, and it's a bit surprising that there're something he still doesn't know about, such as the stationary rule (examinees are not allowed to use their own ones) and the flow the whole test. Anyway, I reckon I've written most of my know-how on the tips.pdf I sent him before. All I can do is just tell him to have a quick review on the tips, take a shower and hit the sack. All the best to you, Jason.

Interloan

To find out what the standard method for Campylobacter is, I need to refer to NCCLS (National Committee for Clinical Laboratory Standards). Due to unavilibility of this item either in our library or online, I've issued an interloan request today. The item is: NCCLS (2002) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A2, 2nd edn. Wayne, PA: National Committee for Clinical Laboratory Standards. Since the average wait for an Interloan request is 2-3 weeks, it's supposed to be here by the end of this month.

2 more WMF vulnerabilities

Two New Windows Metafile Bugs Found (on PCWorld.com) Microsoft Windows GRE WMF Format Multiple Unauthorized Memory Access Vulnerabilities (on BugTraq) Broken windows... :P

Home sweet

Finally come back home after heaps of hassle yesterday... but that was not all. Alan Charito almost lost her camcorder bag and digital camera, which were put on the top of our car when we were in Napier!! Hum, she almost can buy another new one. :P

To Napier...!

Lots of hassle happened today - come across drunk driving, 8 hrs driving (which is supposed to take only 4-5 hrs), small SplashPlanet, looking for motel (all of them had been NO VACANCY) and glumpy Hilary... :(

Stream-cast online!

Possibly because of the age(?), my portable radio is getting under performace. It usually lose its tune so that eventually can't hear any sound but noise. Even if I re-tune it, it lost its sound again as soon as my hand leaves from it. It's useless! I'm picking it's getting sensitive to surrounding noise (any kinds of noise - including light!) Here I have an idea - why not switch to online streaming broadcast since we've got broadband? So that's why I turn my laptop into a "DJ box". Here's the how-to: OS: FreeBSD6.0 Realease Package: xmms-1.2.10_4 (/audio/xmms) Plug-in: xmms-liveice (/audio/xmms-liveice) 1. Install programs # cd /usr/ports/audio/xmms; make install clean # cd /usr/ports/audio/xmms-liveice; make install clean 2. xmms and plugin setting Launch xmms, press Ctr-p and a plug-ins list window shows up. In the tab "Effects Plugins" (I though it would be in "Audio I/O Plugins" but actually it's not) there should be a line says "Liveice 1.0.0 [libliveice.so]" if nothing goes wrong. Now everything's almost ready. 3. Choose channels Here I choose Shoutcast and select some casting servers (click on the "Tune in" buttons) and download their playlists. Their playlists are all uniformly named "shoutcast-playlist.pls", hence I have to rename them into other names so that they wouldn't be overwritten by any others. 4. Enjoy it! Everything's got ready. Now what I have to do is just add those downloaded (and renamed) playlists into xmms playlist and play them as normal musics. Yeah! It rocks!! No more radio disturbance!

5...4...3...

...2...1...Happy New Year to everyone!!! Welcome to the year 2006~~~!!!!