Feeder cell prep

Micromanipulation pipettes

For the type of pipettes, refer to this document available at cookmedical (micromanipulation pipette).

This is the first time I am going to handle the above assembly to manipulate embryos and I am researching the type of micromanipulation pipettes available.

In addition to getting pipettes from manufacturers, these pipettes can be made in the lab using a micropipette puller. Refer to Sutter pipette cookbook to determine the settings required for pulling different types of pipette. These pipettes need to be fire-polished (or flame-polished) to smoothen the surface.

In addition, holding pipette can be generated by briefly exposing micropipette to flame (or via microforge for more precision under microscope observation).

Fig shows fire-polished glass micropipette or pipette. Taken from Nature Publishing.

Fig shows microforge used for processing micropipettes. Taken from Tritechresearch.

A video showing how to process micropipette using microforge and grinder.

Equipment available in the following links

• Narishige needle processing catalog by Tritechresearch
• Sutter Instrument micropipettes fabrication tools.
• Leica DMi8 microscope for micromanipulation.

Solved: How to disable highlighting new programs in Windows 7, Vista & XP

Thanks to HowToGeek instruction, I was able to disable the annoying persistent highlight of new programs installed in Windows 7. Refer to the post here with pictorials.

First, move mouse cursor to taskbar, right click to bring out a menu option, then go to properties. Click on the Start Menu tab. Click on Customize… (to customize how links, icons, and menus look and behave in the Start Menu). Scroll down a list and search for “Highlight newly installed programs”. Deselect that. Done.

Scientific misconduct and recent cases

I followed the first one about a Japanese researcher Haruko Obokata who was suspected of spiking her work with stem cells from other sources to suggest that she could induce inducible pluripotent stem cells iPSCs by stressing normal cells (or somatic cells).

Recently, another case in the US about a researcher Dong Pyou Han who spiked his rabbit blood samples with human antibodies to suggest that vaccine injected to the rabbits was able to induce immune response against the vaccine against HIV.

For the latest case, the perpetrator got a prison sentence.

XIST and pol III in XCI

X-chromosome inactive specific transcript (or Xist) is involved in X chromosome inactivation (XCI).
-
Silencing or kd of Xist in somatic cells only produces low level reactivation in XCI.
-
Those reactivated XCI transcripts are non-coding and upstream of the regulatory region contains SINEs (Short-INterspersed repetitive Elements targeted by RNA pol III).
-
Involvement of RNA pol III?
-
Kd of RNA pol III changed chromatin epigenetic landscape. A promising sign.
-
Refers to work by Prof Zhang Li-feng (Nanyang Technological University)

Mnemonic for nucleotide nomenclature

This information is procured from "Nucleotide Nomenclature and Symbol" (In Biological Sciences).

Mnemonic for nucleotide nomenclature and symbol. The letters cap and underlined are the symbols. E.g. W represents Adenine and Thymine (or W = A or T), K represents Guanine and Thymine (K = G or T).

Information unfriendly commercial companies

I get it, that secret ingredients are what makes commercial companies thrive.
-
Unfortunately, kits with minimal information is not helping students understand the concept behind workability of the kit (or approach). It is a disservice to Science and knowledge.
-
I never liked companies that provided less-to-no information about their products. I shun away from these companies to save myself from being made into a technical person who follow instructions.
-
Some companies are friendlier while others are protective of self-interest.
-
For those who want to learn more about kits, the ideal place to look would be via patent files. Google is one place to start.
-
Just to share.

Estimate the molecular/formula weight of DNA based on its length in base pair (bp)

This is just an estimation, i.e. 650.
-
How to calculate Molarity and Number of DNA molecules

• Calculate the molecular weight of DNA molecule

A double stranded DNA of 100 bp will have an estimated molecular/formula weight = 100 bp x 650/bp = 65,000. Note that 650 is the molecular weight of one base pair.
-

• Calculate the Molarity of DNA solution

To calculate molarity of DNA, e.g. 100 µg/ml (equivalent to 0.1 g/l) of 100 bp DNA will corresponds to 0.1/65000 Molar = 1.5 µM DNA (equivalent to 1.5 µmol/l).
-

• Calculate the number of DNA molecules in a volume of DNA solution

To calculate the numbers of DNA molecules in 1 µl (equivalent to 1 x 10^-6 l) of 1.5 µM DNA, use the Avagadro number to convert mol to the number of molecules. First, determine the mol of DNA in 1 µl of 1.5 µmol/l DNA solution, e.g. 1.5 x 10^-6 x 10^-6 = 1.5 x 10^-12 mol. Next, determine number of DNA molecules, e.g. 1.5 x 10^-12 x 6.023 x 10^23 = 9.03 x 10^11 DNA molecules. That is equivalent to 0.9 trillion DNA molecules.
-

• Calculate the number of DNA molecules based on mass

If I have a mass of DNA molecule, e.g. 135 pg of 500 bp dsDNA, how many DNA molecules is there? First, determine the molecular weight, i.e. 500 bp x 650/bp = 325,000. Note that 135 pg is equivalent to 135 x 10^-12 g. Next, determine the mol of DNA, i.e. 135 x 10^-12 / 325,000 = 4.15 x 10^-16 mol. In a mole, there is 6.023 x 10^23 molecules, thus the amount of DNA molecules is 4.15 x 10^-16 x 6.023 x 10^23 = 250 million DNA molecules.
-
This is for my own reference.

Prevent autoclave water from seeping into pipette tip boxes

When I want to autoclave pipette tip boxes, I would place these boxes at higher level during autoclave. Placing these boxes (or other materials) near to the perforated platform just above the autoclave water would invite the "dirty" autoclave water (sometimes we tend to change the water once every two days or more) from seeping into the tip boxes. The brownish (probably due to spilt media) water will seep into the boxes and when dried, the tips will have a yellowish to brown stain covering them later.

When the tip boxes are placed neat to the perforate platform just above the autoclave water, then during the autoclave, the dirty water will thrash inside the autoclave and some of the water will seep into the tip boxes via cracks. That will contaminate these boxes.

The dirty water might contain impurities that will inhibit sensitive reactions or worst, contaminate stock solutions, enzymes, and expensive reagents.

Not all labs would use commercially packed filtered tips and we do use sterilized tips for dispensing expensive reagents. It is very important to properly autoclave tip boxes so that no contamination from the autoclave water will occur.

I had my fair share of dirty tips due to the above type of autoclave machine.

Just to share.

Inspiring story about Robert Langer

This is a story about Robert Langer,  "Engineer of Change" by TheScientist. 

At times, it's so challenging to stay in Science. If you don't publish, it's difficult to stay on to do research, as you might get penalized during job application or interview (for not having enough publications). I'm struggling with that now and it's demoralizing to get no rejection (no news).

I have to remind myself that it's all about having the right attitude (to get through the day). If indeed there is genuine love and passion for Science and the believe that our contribution will benefit mankind, environment and future, I guess going at different callings (opportunity that came knocking during the downs) will eventually lead us back to Science. For now, I'm applying for more options than just relying on research.

This article helped

DIY Brine shrimp (or Artemia) hatchery

Introduction
Brine shrimp (or Artemia salina) is a crustacean used as a food source for fry and fish. In a zebrafish facility, brine shrimp is used as a intermittent feed for the fry and adults. Shrimp hatchlings at less than two days old contain yolky reserve rich in nutrients for fry and adult fish (feeding them artemia increases better egg production in females).

Materials

• Brine shrimp eggs. Don't get those decapsulated eggs that will not hatch BUT used directly to feed fish instead. For bulk order, you can visit BrineShrimpDirect (I have not dealt with them so any question do contact them directly). In eBay, the cost is  about SGD 2.20 per 10 g of eggs. Normally, I will use 2 tbsp per 1 litre sea salt water and allow it to hatch overnight with aeration.

Brine shrimp eggs from eBay seller fishmanofrobertsbridge.

• Aquarium sea salt mixture (weighing 1.36 kg) available at eBay from timholliday at approx. SGD 36.00 (inclusive of shipment). About 20 to 30 g salt per liter of water is required to hatch brine shrimp (but best would be to follow the instruction from shrimp egg producer).

Sea salt from eBay seller timholliday.

• Shrimp hatchery set. You can DIY by using a Coca Cola 2 liter bottle, aquarium tubing, and plastic hollow balloon sticks (see fig below).

 

DIY brine shrimp hatchery. Instead of having a nozzle at the bottom to drain out the hatched shrimp, I prefer using the balloon stick connected to tubing to siphon off the shrimps from the vessel. That way, I will not be collecting unhatched eggs settled to the bottom as I would by using the bottom outlet (in commercial setting). 

Once the unhatched eggs are settled to the bottom and the empty cyst floating at the surface, I would usually use the hollow stick/straw connected to tubing to siphon out the shrimp (indicated red). A muslin cloth is normally used to net the shrimp and added back to fish water (at a reduced salt concentration) before feeding.

Different stages of maturity of the brine shrimps. New hatchlings are preferred due to their high yolk content, whereas older shrimps might have loss some yolky storage. The unhatched eggs are shown here.

 For commercial hatchery, you can search for them from eBay. Just key in brine shrimp hatchery and you will get returns.

Notes

• Hatching the eggs is good for maximum three days. After that, the shrimp will lose its yolk reserve and would also have lost their nutritious value. If you need to keep shrimp for more than three days, it is best to feed the brine shrimp in order to keep them alive.
• Don't feed too much shrimp to your fish because this food is considered fatty. Feed the fish once per day and add in dry food for the other two feeding per day (total 3 feeding).
• Fish will always be hungry. So, don't allow demand feeding because there will always be demand. Fish will die of obesity-related complications!

DIY material

Hollow balloon sticks (get white ones if preferred). Fig from www.balloonsandweights.com.

Muslin cloth or cloth coffee filter (you can get this from Aliexpress or shops)

In vitro cell culture changes epigenetic marks

This study demonstrates that cell culture could change the epigenetic mark of cells taken from tissues. In this paper, the 5hmC marks reduced drastically after plating mouse fibroblast taken from mice.  The factors involved are reduced Tet protein expression (important for modifying 5- methylcytosine to 5-hydroxymethylcytosine), which is responsive to environmental changes and loss of cofactors important for Tet activity.