Protein concentration, membrane based, Molecular Weight Cutoff aka Nominal Molecular Weight Limit

Protein concentrator based on membrane and centrifugation works by removing biomolecules at determined molecular size/weight. For example, a membrane-based protein concentrator such as Centriprep Centrifugal Filter Unit with Ultracel-50 membrane (Merck Millipore cat# 4311) has a protein cutoff point at 50 kDa. The term used can be  Molecular Weight Cutoff (MWCO; refer to Wikipedia) or Nominal Molecular Weight Limit (NMWL) but they mean similar thing, that proteins at or above 50 kDa is trapped/concentrated whereas molecules at <50 be="" br="" kda="" removed.="" will="">

Abstract

Writing abstract
If writers are able to conceive the detail of the abstract prior to writing other parts of a paper, thesis etc., it will be of great help. Abstracts can be considered as a mini-thesis (at around 100 words).

It should contain the following information:

• What was done/what are the findings?
• The reason?
• The method?
• The results?
• The significance of finding?

References
Writing an abstract (link)

How to go about hard binding thesis in NTU Singapore

At last, finished my PhD defense and on the way to hard bind my thesis. Although NTU Library no longer collect hard copy theses, NTU schools still collect them for archiving. So, I need to prepare these hard copies.

To have my thesis bound in hard cover, I had to do it at the NTU library printing station (Campus Supplies Pte Ltd). The printing station is equipped with plethora of printers, scanners, and photocopiers. The payment method can be made via Ezlink. The shop was well equipped to cater to students, staffs and lecturers' needs.

One thing I didn't really fancy in the place. Not all staff is friendly. They are not rude, but they aren't welcoming and friendly. What a shame. The whole affair was very terse and impatient. Fortunately, not all the staff are like that. I guess their mundane work and the lack of friendlier and conducive environment sort of transformed them into zombie-like.

Anyway, the cost for hard binding a thesis is SGD 18. For two theses, it costs SGD 34. There is a submission date and a collection date (which is a week after submission). The theses are sent to a binder and collected in a week's time.

Alternatively, students can also contact another shop providing the above service, e.g. Ultra Supplies at Queensway. I have not contacted them because the place is far from NTU and probably nearer to students staying near NUS.

Contact
Ultra Supplies
No. 1 Queensway Office, #03-38/39
Main Service Counters, #03-37/45
Queensway Shopping Centre
Singapore 149053
Tel: 64796074
Fax: 64750845

For friendly assistance, call
Philip at 90214846
Kevin at 90216062

Campus Supplies Pte Ltd
Lee Wee Nam Library NTU
Tel: 67935980
Email: campusntu@campus-supplies.com

Mailing address:
Kent Ridge P. O. Box 1053
S911102

If given the chance, I would like to try dealing with Ultra Supplies and compare the service standard with that of Campus Supplies Pte Ltd. This is considering that the business card of the former included the description "for friendly assistance, call". That's a strange way to assure customers of friendly service. I would have expected all staffs should be friendly rather than assigning certain staffs as friendly...

Strange thought.

Ligation independent cloning

I was following Nick Oswald's approach for ligation independent cloning (LIC) until I realized that his method can't really work. The reference to the original work that he linked to was okay and should be adhered to. I think Nick's method failed because he was thinking of adding a start codon and several bases just to bring up the number of bases to at least 15 bases of "overlap" (the N-term amino acid MRRGAP). However, when you studied the bases that he used in the example (I'm guessing that the method he showed was just a proof-of-concept, without any empirical value), one big problem arises.

The %GC content is extremely high (at 78.8% GC). This will proof to be a downfall because whenever users want to design primers that incorporate termini that is complementary to the LIC sequence, they will realize that the homo-dimerization will be a big problem (homodimer delta G = -22.78 kcal/mole). That is just for the forward primer with 5' extension of TGCGGCGTGGCGCGCCGCAA. This alone is already a big headache to many people. Another major problem is that there is a hairpin problem. The formation of hairpin is very likely! Imagine what will happen if more gene-specific bases are added to the bases already prone to hairpin formation...

I don't understand why Nick's approach requires incorporation of start codon. If LIC cloning is really versatile, addition of ATG to the sequence after the LIC complementary termini should be a breeze. 

I have made the vector with the LIC sequence, but I'm afraid I'm stuck with the primer design and PCR steps. I wished I had studied Nick's method in detail before embarking on the DNA vector construction. This entry is just to warn others to read Nick's approach with caveat. My advise is that if you want to incorporate LIC sequence, refer to this paper.

Definition my friend, without definition I can't understand you

In written work, please provide sufficient (if not redundant) amount of definition/explanation of terms used in the writing. If a writer fails to do so, it will be very difficult for readers to understand what you are trying to say.

In a conversation, at least when the listener can't understand a term/expression introduced into the speech, he/she can still stop the speaker on his/her track to ask about the ambiguous or foreign term (or expression) just used.

So, when someone is talking about "input tax", "residual supplies, claim, and "De Minimis Rule", a Science person like me only hear tax input, what supply leftover, claim, minimal rule, what? It make no sense.

How to manually merge two channels using Zeiss Axiovision

I forgets how to do this and thus, having a blog to refer to will be useful. This is a manual method to merge images taken using Axiovision. There is an automatic method (multidimensional) to acquire and merge the images, but I will not cover it here.

First, acquire multiple images with different channel, e.g. Bright field (BF), Green, Red, Blue etc. Then go to the first image and click on Processing in the menu bar, scroll down and click on Add Channel. A popup will show the first image and there is a box to check for [preview].

On the bottom, there is input 1 to input 3 (if you want to merge three images). Click ok and the software will generate the merged image. In order to view the different channels, and merge image in a screen, click on the "gallery view" tab at the bottom of the image. You can individually select a channel and adjust the brightness. There could be other adjustment to image you can do.

That's it.

Cluster of differentiation (CD) molecules; nomenclature and list 2014

The list of CD designation,  nomenclature, and others can be accessed in uniprot (www.uniprot.org/docs/cdlist.txt).

How to elute delivered DNA on Whatman paper

Usually,  we deliver DNA (1 ug) dried on a Whatman paper and the spot is circled with a pencil to mark the spot. That is the most convenient way to post/mail DNA abroad. It's better and more cost-saving than shipping DNA solution in a tube.

Once the DNA reaches the lab,  we would cut out the spot demarcated by pencil and place it in a purification column and elute it with TE buffer.  However,  a purification column costs sgd 1.70 per item (or lesser now).

Another cheaper way to elute the DNA is by using a 0.5 ml tube (with the cap removed) and stack it into a 1.5 ml tube.  Then by means of a syringe,  the bottom of the 0.5 ml tube is perforated by repeated jabbing. Next,  the cut-out DNA on paper is added into the 0.5 ml tube and sufficient TE buffer (e.g. 30 ml) is added to soak the cut-out piece. The assembly is the spun at max speed for 1 to 2 min to elute the DNA solution into the 1.5 ml tube. See figure for reference.

Mnemonic - RNA pol II holoenzyme (my ref)

There are total of six general transcription factors (complexes):

TFIIA (Assist) TFIID to (Dock) to DNA. TFIIB (Binds) to TFIID to recruit RNA pol II. TFIIF (Fidelity) bound to RNA pol II prevents unspecific docking. TFIIE (Elongation) and TFIIH (Helicase) are recruited to docked RNA pol II and prepare from initiation to elongation phase.

"Good Samaritan corner" in the lab

Good Samaritan corner is needed in research lab. In my lab, common reagents/solutions were prepared by a single person and shared by many people. That took a toll on that person (especially when someone later complained about lack of solutions that affected experiments) and since then, there was no longer common reagents/solutions and everyone was supposed to prepare his/her own solutions.

I'm planning to assign a corner (or other places) in the lab "Good Samaritan corner" where we can volunteer to prepare solution without any expectation of its availability. If the solution is unavailable, no one should should, "solution A is empty!". The corner will be "you reap what you sow", in which the availability of common solutions is a shared volunteerism.

I like this advert, " The FLoid® Cell Imaging Station frees students tofocus on cell biology rather than instrumentoperation"

How true.  If only instrument or software platforms can be more friendly to end users that we needn't spend too much time dealing with operation manual and instead go straight down to business of answers.

Items for zebrafish injection

Capillary tube ( www.wpi-europe.com/products/glass/patch-clamp-glass-capillaries.aspx).

Lab cleanliness without broom and dust

Labs are normally cleaned by contracted company and their employees use different methods to remove dirt from the floor. The worst method I've seen so far is by using broom to sweep up the dirt. The smaller particles are prone to linger around the air and subsequently land onto benches, bottles, and opened solutions. If you are plating on the same day the floor is swept, you will realize that the plates will get contaminated with fungi the next day (if you are lucky). That's how bad sweeping in the lab is.

The best way to prevent unsettling dust and contaminating the air in the lab is to use electrostatic fabric that will attract dusts and which can be subsequently disposed of.

Minimum information about an experiment (MIAxE)

Minimum information about an experiment (MIAxE) is a publication standard to provide necessary information for other labs to be able to replicate the published experiment.

There are currently two well-known MIAxEs: Minimum information about qPCR experiment (MIAQE or MIQE) and minimum information about microarray experiment (MIAME).

I hope there will be other standards to facilitate other labs to validate published results in respected journals. There shouldn't be excuses as to why some experiments can't be replicated. Although biological system is unpredictable (sometimes) or variable (most of the time), these variabilities wouldn't be to huge to prevent reproducibility of results.