BioForum

NIH 3T3 cells cross contamination

Tue, 18 Jun 2013 05:42:14 +0000

Dear all,
In our lab we usually keep cells immersed in liquid nitrogen
when I thaw my cells, I found some liquid nitrogen inside the tube.
So I put it in the clean bench and waited till all liq evaporate and melted by room temp to avoid explosion of the tube if I tried to thaw in warm water bath.
and it really worked out, and cells melted without any breakage of tube.

However, I am afraid of cross contamination.

I have this threat inside my head < perhaps other cells are just like these tubes and cells my transferred from one tube to another .
all NIH 3T3 tube contain the same problem, I checked our stock, all tubes has LN leaked inside the tube,
and I could not check all other cell tubes as the container contains other people work too., and a lot of tubes
any advice about this threat???
Thank u
madelin

NIH 3T3 cells

Tue, 18 Jun 2013 05:31:29 +0000

Dear All,
it is my first time to use NIH 3t3 cells for the formation of native Extracellular matrix

I have some questions?
at first: I read some protocol about maintaining it Calf serum with high glucose DMEM.

But I find another website suggest 10 % FBS with DMEM (not high glucose)?
usually in our lab we use heat inactivated FBS, so I just thaw my cells in 10% heat inactivated FBS in DMEM.
I will try to see if it will effect Extra cellular matrix secretion or not but do u have any suggestion??

The second thing, they said never leave it till become 100% confluent, but I really left it, yesterday it was almost 70%, but I said leave it till tomorrow as I wanted to make alot of stock?
so today it was almost 100 % confluent??
how does this affect my cells.
Please if u have further comments regarding this type of cell, kindly share it with me.
Regards
Madelin

PCR to get 10kbp product

Tue, 18 Jun 2013 04:48:00 +0000

Hi all,
I have to get 10kbp PCR product.
I have 2 primers, which have Tm of 58oC (F) and 60oC ®. I intend to use Pfu Taq pol to get the high accuracy.
What should I do to get success?
Thank you so much for your suggestion!

Promoter distance from ATG

Tue, 18 Jun 2013 00:11:58 +0000

Hey all,

I'm planning to clone a fairly complex construct for conditional shRNA, and one step is to include a tissue specific promoter 5' of the whole thing. I've never had experience placing promoters far from the start site in transgenic constructs. How far away can one place a promoter (in this case the Gfa2 glial-specific promoter) from a downstream CDS start site?

There simply aren't many restriction sites in my vectors that would allow me to get closer than ~250bp. As it stands right now, the cleanest/most efficient way to insert the promoter sequence would leave a gap of 277 base pairs. Will this be sufficient to drive expression?

Any advice is appreciated, I'm fairly new to this.

Thanks

site-directed mutagenesis primer Tm

Mon, 17 Jun 2013 20:24:12 +0000

hi everyone,

i'm using partially overlapping primers so the chance for primer dimerization is eliminated. i was wondering if the recommendation of the Tm difference not larger than 5 °C still applies for site-directed mutagenesis primers. or if it even might be better to use primers with about the same Tm. does anyone have some more solid information about this?

bentham science publisher

Mon, 17 Jun 2013 17:23:44 +0000

Hallo all,

someone at the lab has the possibility to publish in one of bentham science journals.
Now I am wondering: should this be done?
My name would be on it too and I have something to say abou it too and I am a bit reluctant to publish in one of those journals.

I also find it strange that pretty much no university on this planet has acces to their journals (except the open one).

I have read some negative points about this publisher so I think its best not th publish in it?

Anyone here published in it?

Testing nucleotide-protein interaction

Mon, 17 Jun 2013 16:28:37 +0000

Hi everybody

I work with an intrinsically disordered protein with unknown function and no close homologs. Psi Blasts show some low degree of homology to ATPsaes, but with e-values above cutoff. Motif searches predict an incomplete Walker A motif. If so, only the GKS motif is conserved, but not the P-loop.
The protein forms a stable complex with Ca-calmodulin.

I tried atpase assays, but there was no activity. Upon addition of Mg-ATP and Mg-GTP there is an increased precipitation of the complex with increasing GTP concentrations, but not with ATP (I checked the pH of both solutions).

Running a native gel with lower GTP/ATP concentrations does not result in a visible gel shift. I tried several other nucleotides, but without result.

Could you give me some ideas how to test the function/nucleotide binding abilities of this protein? Is there a change that this protein is a nucleotide binding protein at all?

Thanks

Membrane protein

Mon, 17 Jun 2013 16:27:01 +0000

Hello everyone,

I am working on a specific membrane protein. Any suggestions as to which expression host would be best for such proteins (bacteria, yeast) etc.

Thanks.
Manavid

Undergrad "activated" a NS membrane with 100% MeOH... Any hope?

Mon, 17 Jun 2013 14:52:25 +0000

Hey all,
So the undergrad tried to activate a nitrocellulose membrane with 100% MeOH. Is there any hope of getting an actual result with this now?

Thanks in advance for your help!

sequencing

Mon, 17 Jun 2013 14:42:18 +0000

Hello All,

I need a help from you.
the question is i using a cell type XXXX and i want to check the genotype regarding this SNP in the genotype.
so for this can i extract dna from these cell type and can i use this DNA to sequence instead of doing pcr?

Hope i am clear.

Thanx in advance

Harry

Vesicular Monoamine Transporter2 (VMAT2)

Mon, 17 Jun 2013 14:39:51 +0000

Hi,
I have been doing a lot of Western Blotting for the protein Vesicular Monoamine Transporter2 (VMAT2). I'm currently using a rabbit polyclonal antibody from Millipore. The antibody is very temperamental. Sometimes I get a single band and sometimes multiple bands. Many times I get no signal at all. I keep the experimental conditions same all the time. I even tried making samples without beta-mercaptoethanol and even without boiling. But I can be never sure that I will get any signal. Are there some specific conditions this protein needs to be processed? Or is there a better antibody for this? Please help

Trouble with shRNA transfection

Mon, 17 Jun 2013 11:26:59 +0000

Hi everyone,

I need some help about shRNA.

Here is my story : I ordered shRNA plasmids from a commercial firm ( don't know if I can tell the name without being kicked of the topic...) to invalidate the receptor I'm studying. I made amplification and purification, then digestion to linearize the plasmid and transfected it in the cells.
After 24h-48h of rescue, I started a selection with puromycin at the minimal letal dose (I made a killing curve before, and obtained 0,1 µg/mL for one cell line and 1µg/mL for another). I had a massive death of the cell during one week, then growth of resistant clones.
I made a FACS analysis to compare the "target sh" versus Non" target sh" cells.................. and they have the same rate of expression, i.e no invalidation....The cells grow well in puromycin....
Does someone ever face the same problem?
I just see two possibilities : either I had no luck and selected "natural" resistant clones in the culture, which do not integrate the plasmid or the shRNA is not efficient (but I think the firm has validated the sh.....)

bye!

Measuring Fluorescene of RFP

Mon, 17 Jun 2013 05:19:27 +0000

Hello,

I was wondering if someone with more knowledge than me in the area of measuring fluorescent intensity of fluorescent proteins can shed some light on this new topic. I am a newbie in this field so I don't know precise terminology but I will try to make sense:

I have transformed bacteria containing a plasmid with a promoter-RFP (red fluorescent protein) fusion that I am using as a reporter of gene activity. The RFP is dsRed from dsRed-Express2 plasmid, and it has an excitation maximum of 554nm and an emission maximum of 591nm. My question is do I need to set the excitation and emission values exactly on the machine (a PerkinElmer Victor x5 multilabel plate reader)? Is there one that I must set exactly right?

We currently don't have filters that exactly match those numbers so I am running my experiments without them but I am not getting good results. I wanted to know if the settings were the problem or some other aspect of my experiment may be it.

Thank you,
Ted

Genomic DNA and plasmid DNA isolation

Mon, 17 Jun 2013 01:12:58 +0000

HI, i will be doing whole genome sequence of a bacteria. And for that i need gDNA and plasmid both in the same isolate. I am using Promega DNA purification kit for DNA extraction. Is this kit able to isolate bothe gDNA and Plasmid in a good amount. Is there any other, method for isolation. Please suggest.
Thank for the co-operation.

Advanced Algal Technologies Limited helped in commercializing

Sun, 16 Jun 2013 10:33:41 +0000

There are many technology providers in Biotechnology Industry. Those company which are focusing on the production of industrial biomass production using patented technology which helps it in commercial production of biomass. Through This Process Helps its user companies to reduce costs through Carbon emission abatement and simultaneously facilitates a commercial revenue stream. Their Technology systems is a low-cost, extensive open pond raceway system designed for producing protein rich content for livestock feed industry. It has varied application in industries pertaining to animal feeds, health food sector, syngas and co-generation electricity, etc. Advanced Algal Technologies Limited is one of those technology providers.

Getting Bacteria from an OD0.8 to an OD 0.1

Sun, 16 Jun 2013 03:06:51 +0000

Hi there,

Say I have 50mL of bacteria at an OD0.8 and I need to get bacteria to a reading of 0.1..this is what I've been doing in the past but just want to confirm that what I've been doing in correct.

So from my 50mL, I took 1mL, spun and then resuspended the pellet in 1mL of PBS. (I now have 1mL of bacteria at OD0.8 in PBS)
I took 125uL from this and added to 875uL of PBS (OD has gone down 8x now)

Is this correct--would this now give me 1mL of bacteria at an OD0.1?

Thanks,
biology_06er

Smallest possible insert size for Ligation reaction

Fri, 14 Jun 2013 23:45:33 +0000

Hi All
This would be my very first post on this nice forum. I hope for some nice and helpfull suggestions and experiences. I was wondering if somebody could share his/ her experience with me about, what can be the smallest possible size of an insert which can be succesfully ligated into a very huge plasmid (around 17 kbp), and is there is any scale for the size of the insert and the size of plasmid for a better ligation reaction?

Thanks all in advance.. looking forward to hear from you.

Transwell migration assay (Boyden chambers) and DAPI staining

Fri, 14 Jun 2013 19:45:38 +0000

Hi all!

Does someone have any experience with the boyden chambers?

I performed this assay and, in the last step, I stained the migrated cells with DAPI after fixing and permeabilizing them. I looked at them with the fluorescence and I could clearly see the blue dots of the nuclei. The problem is that I could not see cells with the bright field, so I am afraid that what I observed could be the aspecific of the membrane pores. Could this be possible? Should DAPI stain only the DNA ? Could the blue dots be migrating cells that are still stacked into the membrane pores?

Thank you very much for your help!

No tubulin detected in HL-60 lysates (???)

Fri, 14 Jun 2013 18:54:21 +0000

Hi All,

I've been working on some experiments in HL-60 cells. Surprisingly, I tried using tubulin as a loading control for my immunoblots, and nothing was detected! (However it works fine in other cell types run on the same blot.) Actin seems to work fine, thus finding a loading control is not the issue. I'm wondering if anyone else has observed this, and what reason(s) might help to answer the case of missing tubulin.

Thanks!

Single-step nested PCR: how to investigate dynamics?

Fri, 14 Jun 2013 10:39:47 +0000

Hi,

I've designed a single-step nested PCR in which two sets of nested primers amplify an outer and an inner amplicon (see attachment). By using a lower inner primer concentration I obtain a large majority of outer amplicons.I'm an now interested in the dynamics of this PCR. I would like to see when the outer primers actually start taking over the amplification from the inner primers and see what influence primer concentration, Tm etc. have on this reaction.

I thought of using probes which are specific for the outer amplicon and one which is non-specific in order to calculate a ratio of each amplicon after each cycle. But designing a specific probe for the outer amplicon proves difficult. Does anybody have an idea how to solve this?

Regards,

Bram