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AussieUSA's Content

There have been 22 items by AussieUSA (Search limited from 15-July 18)


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#86208 health insurance

Posted by AussieUSA on 08 September 2010 - 04:40 AM in Venting and Counseling

Hi Samita,
I am an Aussie who postdoc'ed in the US. With every job I had while on a J2 visa, the employer (university) subsidised my health insurance ... it was part of the package. I later became a permanent resident and was granted a postdoc at the NIH. There my health insurance was paid in full. If I went into a full-time employed/academic position, then I would have had to pay for my insurance, through my employer.

Does this help?

AussieUSA.



#57116 Easy primer question?

Posted by AussieUSA on 29 January 2010 - 02:37 PM in PCR, RT-PCR and Real-Time PCR

Simple answer ... No!

Just quote the paper for sequence info.

Hope this helps,

AussieUSA.



#57114 always run internal control for different genes on the differnet plates?

Posted by AussieUSA on 29 January 2010 - 02:35 PM in PCR, RT-PCR and Real-Time PCR

Everytime you run a plate, there could be a slight difference in amplification based on your pipetting ability, the temperature of the room, you name it. Therefore you should always run the standard on every plate you amplify.

As Stylo states ... a 384-well plate could save you for space.

Have fun,

AussieUSA.



#57112 Reverse transcription question

Posted by AussieUSA on 29 January 2010 - 02:30 PM in PCR, RT-PCR and Real-Time PCR

I know that standardly we use a 20 l reaction volume for second strand synthesis of total RNA using 1-2 g RNA but does anyone routinely use higher reaction volumes? Do you still get adequate results??

I am asking because I have very limited RNA in high volume and have way too many samples to concentrate. If possible, I would like to use a 50 l reaction volume using 1 g RNA. Just need someone else to say "it'll work" and then I'll feel more confident :wacko::-)

AussieUSA.



#57109 stiching/linking/sewing pcr

Posted by AussieUSA on 29 January 2010 - 02:18 PM in PCR, RT-PCR and Real-Time PCR

Also ... increase the time for amplification to ensure products get made and can come together :-)



#57108 stiching/linking/sewing pcr

Posted by AussieUSA on 29 January 2010 - 02:17 PM in PCR, RT-PCR and Real-Time PCR

I haven't done this from genomic DNA but this could work ... start with the annealing temperature low (not too low) to ensure some production of amplicons then every 5th cycle, raise the temperature in increments so that the specificity is increased. It is possible that in the genomic DNA the spacing is too far to allow the amplicons to come together. You will need to gel purify to ensure you clone the product of interest.

Hope this helps,

AussieUSA.



#57107 DeltaDelta Ct method and Statistics

Posted by AussieUSA on 29 January 2010 - 02:13 PM in PCR, RT-PCR and Real-Time PCR

If you perform 3 PCR runs then you will have 3 replicates in which to compare.

hope this helps,

AussieUSA.



#38816 Lab Member sabotaging other postdocs experiments

Posted by AussieUSA on 05 October 2009 - 10:04 AM in Venting and Counseling

One proven instance of #2 or #6 should be grounds for immediate firing and perhaps other legal actions to protect the institution and the grantee.
The rest of them are just the guy being a PITA. He should be counseled by the PI to discontinue these practices and, if he doesn't, then he should be fired.



We were never able to fully prove #2 but the postdoc was routinely seen handling our PBS bottles (they were stored in a different part of the lab to his stuff). And two staff members independently spoke to the boss regarding #6 over a 2 year period prior to submission of the paper. One staff member was the postdocs' trainee and when he mentioned his concerns to the boss, the boss was furious and made it very hard for the trainee in the lab thereafter. He eventually left the lab. I was the other staff member, and every time I raised a concern, I was told to keep quiet and as time went on, certain priviledges were denied to me. One suggestion I had was to have an ombudsman review the issue so the boss did bring in an "independent reviewer" but as they were never told what the concerns were, they never knew what to ask or look for. I was informed the independent reviewer found no issue.

The fact that the boss was so <can't use that word here>, is what is spurring me on to have a policy in place at my work so this doesn't happen again (although it seems to happen alot). Rant rant rant ... sorry :-(



#38815 unprepared labmates

Posted by AussieUSA on 05 October 2009 - 09:49 AM in Venting and Counseling

If I had my way, these type of people (lazy, incompetent &^%%*) would become human lab rats (i.e. we inject and study them).

I know every lab has one, at least every now and again, but why do we tolerate them? They have no concept of safety or show no respect to their fellow colleagues. Precious lab resources (like money) just gets wasted and bosses/lab managers just go ... "oh well". And after 20 years in science, I can vouch that not everyone like this leaves science ... they just find labs that leave them be.

I like the idea of emailing them about the issues while cc'ing the boss. Then if nothing changes, you can send a copy of the emails to the next person in the hierarchy etc. until the message gets across.

I also want to ask ... is this really an "MD" and/or "Chinese" thing? I agree that it is but is it really that universal?

Keep sharing Toejam :(

AussieUSA.



#38813 Vector NTI from Invitrogen

Posted by AussieUSA on 05 October 2009 - 09:29 AM in Molecular Cloning

Vector NTI is a great program. It is very similar to MacVector but for the PC not the Mac.

The only annoying feature with Vector NTi, which may no longer exist, is that when I last used it, you could not visualise the electrophograms and modify sequence data generated from the ABI sequence analysers, which can be easily done by MacVector. We used software that used to cost $60AUS to generate the electrophograms and modify the sequences then import into Vector NTI.

I hope this helps,

AussieUSA.



#38812 PBS or TBS

Posted by AussieUSA on 05 October 2009 - 09:22 AM in Protein and Proteomics

Although some people say PBS will interfere in the specificity of the phospho-specific antibody, it is really dependent on your antibody. If the TBS is causing problems, consider adding tween 20 (0.05%) to the TBS, to help eliminate background problems.

Ultimately, do what works for you and your western.

AussieUSA.



#38810 Filtering cytokines??

Posted by AussieUSA on 05 October 2009 - 09:18 AM in Tissue and Cell Culture

I wouldn't recommend added the cytokine to the main media bottle before filtering. You may loose some of the cytokine by filtering but more importantly, you may loose activity if the cytokine is diluted for too long a period of time.

If you are only adding the cytokine to your cells for up to 48h, it is not crucial that it be 100% sterile. Most cytokines come "clean" but never 100% sterile to begin with.

Hope this helps,

AussieUSA.



#38804 Strange lane pattern, yes/no pattern

Posted by AussieUSA on 05 October 2009 - 08:05 AM in SDS-PAGE and Western Blotting

If you are talking about running a protein gel, one of the problems could be that the treated samples are more concentrated than your normal samples or a component in those samples is more concentrated, causing those lanes to bulge out.

Are you loaded the same amount of protein per lane?

AussieUSA.



#38799 Lab Member sabotaging other postdocs experiments

Posted by AussieUSA on 05 October 2009 - 07:23 AM in Venting and Counseling

I also face the similiar problem in your list 1, 3, 4, 5. I also got no idea how to deal with her.
"SOMEONE" had purchased some labwares with our group's research fund, hide it and even denies it, centrifuge when there is a broken tubes contain possible airborne bacteria. "SOMETWO" don't know how to use equipments but use it without asking. "somethree and four" hogging the PCR machine... "Somefive and six" hogging the laminar hood... "someseven" even start a so called "ISO" system and begin recording whatever people do....

When there is more and more people in a lab, there are more politics and problems came out.

I really wonder why THAT supervisor wanted to take in so many ppl when there is limited facility...now seems like everybody have to queue for their turn. Imagine, today I run my pcr in the morning but I have to wait till tomorrow then only I got chance to cast and visualise my gel, not forget to mention there is not enough gel tray to do my agarose gel, PCR machine have to be pre-book 3 days before...
:) :huh: :angry: :(


I am sorry to hear this Adrian.

I too have worked in crowded busy labs like this ... one answer is to work shifts, if that is a easy option for you. When real-time PCR first came out, our lab had one of the first optical machines and it went 24h a day ... that meant us, the users, where in setting plates up every 2-3h. Some people liked to work nights and others preferred early mornings. I am not suggestion you work at midnight, but maybe choose a time when the other lab members are not around as much.

AussieUSA.



#38798 Lab Member sabotaging other postdocs experiments

Posted by AussieUSA on 05 October 2009 - 07:18 AM in Venting and Counseling

If I read it, I would be afraid to even start working at certain labs.

I can not understand that some people out there are doing this.

And I wonder: how do you even know he or she is doing this?

How do you even know he is doing this?

The first incident = In September each year (end of financial year in US), our lab has an ordering freeze for 2-4 weeks while the accountants do their thing. In mid-August 2005, my first year here (and the perpetrators first year here), I took a stock take of our cell culture transfection reagents and media etc. and found we had sufficient reagents for everyone in the lab for 2-3 months so the decision was made by all lab members that we would not need to order these reagents. The first day the ordering freeze took affect, my trainee went into the cell culture lab to start a transfection and the transfection reagent was gone. Everyone denied knowing what happened and we were all stumped. 2 weeks later, after I had reorganised our work plan, we decided to clean out the cell culture fridge (4˚C) and found some tube storage boxes "built" into a cubby hole. Inside this hole, we not only found the general vial of transfection reagent but a new pack of 5 vials marked as received in early August by the perpetrator. Prior to this, the reagent was always kept on the door of the fridge, nowhere near the storage boxes. We showed the lab manager and she approached the guy. This is how it went ... Q1: have you been able to do your transfection experiments over the last 2 weeks? A1: yes. Q2: Which reagent have you been using? A2: Mine. Do you know why the general reagent was hidden behind some storage boxes? A3: No. Q4: But it was hidden next to your reagents. You do not remember seeing it? A4. No. Q5: Did you hide it from the other members? A5: No.

An second example = He recently left the lab (we were very joyous :) ) and he was forced to clean out all his reagents etc. While he was throwing stuff into the trash, we found all the reagents that had gone missing over the years including all the stuff he had denied knowing anything about. Some he had obviously been using and some he had just been hiding. After he left, we did a lab clean up and then found all the labware that had disappeared, in his drawers and cupboards.

So basically, he was "caught red-handed" yet always denied it and the rest of us were told ... it must have been a misunderstanding.



#38450 Drugs first and then cytokines, or at same time?

Posted by AussieUSA on 01 October 2009 - 11:14 AM in Tissue and Cell Culture

Jakatta,

welcome to the research world of immunosuppression, where some labs add stimulant and immunosuppressant at the same time, some add the immunosuppressive agent first (1-12h before) and some add after the stimulant (30-12h).

I too have asked ... why add the anti-inflammatory agent prior to the immune stimulant since this is not the order that occurs in the body. The answer ... it is the way it is done.

CellSpecific is also right ... do as the PI says.

My solution ... if not too hard, try all variations and see what you get.

Also, are you looking for an RNA response or protein response? How long is your treatment time? This could also be a factor as protein may need 6-24h treatment before you can measure a response, and some genes are "slow".

Sorry for not being able to give a concrete answer but enjoy.

AussieUSA.



#38439 Lab Member sabotaging other postdocs experiments

Posted by AussieUSA on 01 October 2009 - 10:12 AM in Venting and Counseling

After spending 4.5 years with a fellow (M.D., Ph.D.) who did everything possible to prevent other post-docs in the lab from getting good data and then having the boss (an M.D.) say "he wouldn't do that, he has an M.D.", I was actually wondering how common it was that one member of a lab would sabotage other members experiments.

Some examples of what he has done include:
  • hiding common reagents and labware then denying he knew anything about it
  • adding detergent to other peoples PBS used in cell culture thus leading to cell lysis
  • breaking equipment and not telling anyone
  • taking and using reagents you were using for a particular experiment and not replacing them
  • informing students assigned to him not to talk to other staff members (his two students quit before their time)
  • forging data for a paper (his only one)

I have noticed that some graduate schools in the US are initiating a policy where these things are termed Academic Sabotage. If you are proven to have done these things, you are out.

Have you experienced this? Did you talk to boss and/or superiors? What was the outcome? Does your lab/institute/university have a policy for this type of behavior?

Please share your thoughts because I would like to try an initiate a policy in my workplace to prevent this from happening. The distrust and low motivation this lead to in the lab turned what once was a fun lab into an awful lab to work in. The fact that the boss ignored us and the fact that going to superiors was considered an incredibly insult to the boss just made it worse (hence the low morale).

Thanks,

AussieUSA.



#36852 Waste bottle

Posted by AussieUSA on 18 September 2009 - 10:47 AM in Tissue and Cell Culture

Dear OK13,

these are very interesting questions and concerns. With regards to the waste bottle, there is no problem with it being outside the hood, and with the tubing stopping the hood from being sealed. Just make sure you dust the area under the work surface every now and again.

If you are concerned about the suction tube being a source of contamination, many labs store the tubing outside attached to the side of the hood. They rinse the outside of the tubing with 70% ethanol before placing back into the hood and using and there are no problems with contamination. I have also seen laminar flow units with an outlet on the side that you can attach "internal" tubing and then there is an outlet for attaching the tubing that connects to the waste bottle. Does your hood have this?

Keeping things inside the hood (waste bottle, partially opened tube bags etc) is a semi-personal thing. In my experience, labs where this is done have more contaminations than labs where everything is removed, allowing complete disinfection of the work surface. In labs where you need GMP, you are not allowed to store anything in the hood. I have also seen disgustingly messy cell culture hoods where people never see contaminations and then I have seen exceptionally clean hoods where staff get all sorts of problems. This could be related more to antibiotic use than "good" asceptic technique.

Let me know what you do and how you succeed in changing things.

AussieUSA.



#36838 Homogenizing animal tissue to RT-PCR in real-time

Posted by AussieUSA on 18 September 2009 - 07:11 AM in PCR, RT-PCR and Real-Time PCR

Similar to Mighty Mouse, we add the tissue to RNAlater, but quick-freezing tissue is fine. Homogenize either in Trizol or any commercially available RNA prep kit (we use Qiagen RNeasy). I might recommend DNase-treating prior to preparing cDNA as tissue is more "messy" than cell line. And treat as normal after that.

Hope this helps,

AussieUSA.



#36836 what is the point of serial dilution???

Posted by AussieUSA on 18 September 2009 - 07:06 AM in Cell Biology

Iamdhan,

are you asking about serial dilution of the cells or serial dilution of the selection agent (antibiotic)?

If you are talking about the selection agent, you do a serial dilution when you do not know the correct concentration of agent to effectively kill off the untransfected cells while keeping your positive cells alive. You only need to do this once in order to determine the concentration to use in subsequent transfections.

I hope this helps,

AussieUSA.



#36833 Do I need to dilute my mouse tissue lysates for micro-BCA?

Posted by AussieUSA on 18 September 2009 - 06:51 AM in Protein and Proteomics

I should mention that I use 3 ml RIPA buffer per gram of tissue :(



#36742 Do I need to dilute my mouse tissue lysates for micro-BCA?

Posted by AussieUSA on 17 September 2009 - 11:47 AM in Protein and Proteomics

Dear Lab Rats,

I have been preparing total protein lysates from spleen, liver and kidney (from mouse) and would like to know should I dilute the samples before adding to my micro-BCA assay (plate method)?

I am usually working with cell lines and dilute from 1:20-1:100 but I know my cell numbers in this situation.

I am hoping to be able to do the assay as a one off, instead of doing multiple titrations ... :)

Thanks for your suggestions,

AussieUSA.




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