Friday, October 7, 2011

MS and PhD studentships in Korea (updated for Fall 2011)

in Chemical Engineering
Excellent opportunies for the Young Graduates


Write to Prof. JJ Shim
@ THE INSTITUTE OF CLEAN TECHNOLOGY
YEUNGNAM UNIVERSITY
_____________________________________________________________________________

214-1 Dae-dong, Gyeongsan, Gyeongbuk 712-749, KOREA
Tel : (53)810-2587 Fax : (53)810-4631 E-mail : jjshim@yu.ac.kr


School of Display and Chemical Engineering at Yeungnam University located near Daegu, the 3rd largest city in Korea, offers full scholarships to young graduates who want to study their MS and PhD in research.

Area of study: Polymers, Organic Chemistry, Inorganic Chemistry, Nano Technology, Clean Technology, Catalysis, Process Control, etc.

Yeungnam University Chunma Art Center

Graduate Research Program


Requirements:
(1) BS and/or MS degree in Chemistry, Chemical Engineering, Biotechnology, and related area (including current UG and MS students who is graduating in Fall 2011.)


(2) English score of TOEFL or IELTS


(3) Academic Record: First class or GPA 3.0/4.0 or above


(4) Research exposure (Not mandatory).



For detail information please download
1. Detail information and requirements


2. Vacancies and Student information
Informal inquiries could be made to Dinesh Thapa, thapa@uthscsa.edu

Tuesday, September 1, 2009

Scholarship in Biotechnology (PhD)

Yeungnam University

School of Biotechnology

A full studentship is available at the school of biotechnolgy, Yeungnam University, Korea. The scholarship covers full tuition waiver and a full maintainace allowances.

Applicants must have (or expect to be awarded) first class MS degree in related fields. They should send a cover letter and current CV for detail information and inquiries, mail to pratistar@yahoo.co.uk

Informal inquires could be made to dinesh@ynu.ac.kr

Friday, August 21, 2009

Lentivirus transduction for stable knockdown of STAT-3

Biohazard: Should be treated as Risk Group Level 2 organisms for laboratory handling.

All handling, storage and disposal of biohazard waste must be in accordance with University rules and regulations.

Materials:
1) 48-well Tissue Culture Microplate
2) Puromycin (Sigma)
3) Hexadimethrine bromide (Sigma)
4) Target Cell Line (MCF-7 and MCF-7/Adr breast cancer cells)
5) DMEM Media
6) FBS
7) Penicillin-Streptomycin
8) 50ml Falcon Tubes
9) 15ml Falcon Tubes
10) 1.5ml RNase Free Tubes


Mission® shRNA Transduction Particles:
1) Non-target control (scramble) transduction particles (Sigma #SHC002V)
Virus titer (Lot 1201010801MN): 1.1×107 TU/ml
2) STAT3 transduction particles (Sigma # NM_003150)
Virus titer: >1×106 TU/ml
Target set (5-clones)
A) TRCN0000020839
B) TRCN0000020840 (validated info => 61% in A549 cells)
C) TRCN0000020841
D) TRCN0000020842 (validated info => 76% in A549 cells)
E) TRCN0000020843 (validated info => 72% in A549 cells)

300 µl/well in 48 well plate
MOI = 1---> Non-target 842 843 Pool (842+843) Hex. Control cells
MOI = 2---> Non-target 842 843 Pool (842+843) Hex. Control cells


Notes:
Every 48-well plate experiment should include:
1. Negative control-Non-target control well (in duplicate)
2. Each clone of shRNA contruct well (in duplicate)
3. A hexadimethrine bromide control only (in duplicate) to determine cell sensitivity
4. A control well with no infection (in duplicate)

Before Starting:
1) Make DMEM media with 10% FBS and 1% PS

2) Prepare a Puromycin stock at 10 mg/ml with sterile water. Aliquot into 1.5mL e-tubes, and store at -20°C.

3) Prepare a hexadimethrine bromide stock solution (2 mg/ml)with sterile water/or media. Add to media to final concentration 8 µg/ml, and filter through syringe filer.

Procedure:

Day One:
1) Cell seeding 5×104 cells/cm2 (/well) in a 48-well plate
70% cell confluency during the day of transduction
Medium for cell seeding: DMEM containing 10% FBS, 1%PS
2) Incubate in a 37ºC incubator at 5% C02 overnight undisturbed.


Day Two:
1) Thaw lentivirus at room temperature (from -70ºC freezer).
2) While virus is thawing, warm DMEM supplemented with 10% FBS and 1%PS.
3) Aspirate the media and replace 200 µl fresh medium containing 12 µg/ml hexadimethrine bromide (final conc. 8 µg/ml in 300 µl).
4) Once the virus is fully thawed, transfer the proper amount for infection to a fresh 1.5ml e-tube containing DMEM to give 100 µl mix per well.
5) Add 100 µl of media containing virus to the cells.
6) Gently mix by swirling the plates and return back to the incubator.
7) Check cytotoxicity after 4 h by observing cells under microscope and change fresh media when toxicity occurs, if not incubate 18-48 h.
Notes:
 An infectious viral titer needs to be determined prior to transduction. Add proper MOI (See Sigma protocol for calculations)
 When transducing a lentiviral construct into a cell for the first time, a range of volume or MOI should be tested. 2,5,10 and 15 ul of lentiviral particles per 1.6 × 104 cells or MOIs of 0.5,1,2, and 5 should be used to determine the optimal transduction efficiency and knockdown for the each cell line.
 You do not want to disturb or stress the cells. Be careful when adding the media.
 Be sure to mark which wells in the plate have received which shRNA.

Day Three:
1) Remove the virus media and replace with DMEM media supplemented with 10% FBS and 1% PS.
2.) Place plate back in the incubator and incubate at 37ºC.

Day Four:
Observe the cells under microscope.
Aspirator the old media and replace fresh media with 500 ul of DMEM supplemented with10% FBS, 1%PS and the proper concentration of Puromycin (according to Kill-curve).

Day Five:
1) If cells are confluent, scale up by passaging to 6 well plate (10 cm2).
2) Continue adding puromycin 1 µg/ml.

Note: The concentration of Puromycin is going to be different with each cell line as some are more sensitive to the drug than others. It is crucial to do a kill curve with each cell line and pick the lowest concentration of Puromycin that will completely kill non-selected cells within 7-10 days.
 MCF-7 cells need 0.5 µg/ml Puromycin whereas MCF-7/Adr cells require more than 5 µg/ml.

Two Weeks Later:
1) Watch the cells carefully over the next 2 weeks, passaging the cells and changing the media as necessary.

NOTE: Be sure each media change or passage contains Puromycin. Once the cells are recovering well from the Puromycin selection (depending on transduction efficiency, many cells may be killed off and it can take a while to grow the transduced cells), you may transfer them to a the culture flask/make lyophilized stocks for further propogation.

2) Confirm your stable cells for particular or known target gene/protein expression by
a) Extract RNA from cells and run RT-PCR experiments.
b) Or Extract cellular proteins and perform Western blot.


Good Luck!!!

References:
1. Dharmacon
2. Sigma
3. Internet references

Thursday, August 20, 2009

shRNA lentiviral particles

Lentiviral particles are widely available for targeted gene knockdown. Recently, I have set up gene knockdown experiment using Mission lentiviral particles from Sigma. I will share this information and basic protocol in next blog.

Tuesday, November 4, 2008

Gene silencing and siRNA

"Once a protocol is optimized for a particular cell type, reproducible siRNA screening experiments can easily be done".

Low transfection efficiency and low cell viability are the most frequent causes of unsuccessful gene silencing experiments. Through careful optimization--e.g. choosing the right transfection agent and transfection method--high levels of transfection efficiency can be achieved in many cell types.

Step1. Select Transfection agent and amount, cell density, and cell type.

Step2. Monitor cell viability and measure target gene knockdown .

Read some of the manufacturer's link for helpful guidelines.

Dharmafect click here

Qiagen click here

Sigma click here

Ambion click here

Friday, October 31, 2008

Oral administration to rats

Are you guys doing some experiments really aware with the facts that what and why are you doing so?? Most of us say/answer- YES, but actually NOT. Among many options, I prefer to visit BioForum, the page crowded with talents and more talents.

Anyway, I was curious to know about Oral administration and some basic fact. Here is one good answer by SamOH.


""Hello,We do a lot of oral gavage administration to mice in the context of experimental anticancer therapeutics.Appropriate volume?: We give 10 microL/gram body weight (i.e. 10 ml/kg) to our mice. The most common volume guideline for rats that I've seen is 20 ml/kg as a maximum. Thus, a 200 g rat would get no more than 4 ml of drug. The idea is to give a volume that is not so large that it forces open the pyloric sphincter or causes reflux into the esophagus. Your local IACUC or lab animal vet would be a good source of info, since it is their guidelines that you must follow.

Moreover, we dose based on the individual animal's true body weight (BW); i.e. we measure BW once/week and then adjust dosing volume for each animal accordingly. Alternatively, you could dose the animals based on the average BW in the group. The drug dose will vary slightly among animals, but it may be easier in that you will administer the same volume to each animal.

Which solvent/vehicle?: The selection of vehicle/formulation is an entire field unto itself of which I am not an expert. My impression is that, if the agent is water-soluble, then any of the 3 you mentioned will work - I've seen all 3 used in the literature. Our agents are generally very hydrophobic, so we dose as a suspension in a methylcellulose/Tween 80-based vehicle. Others have used PEG and cyclodextrin (CD). Tween (polysorbate) is very safe for the rodent; PEG can have toxicities at higher doses; CD may reduce absorption of the drug if it complexes too tightly with the drug. Hope this helps,SK""

Source: BioForum

Monday, October 27, 2008

DNA transfection using Lipofectamine

Efficient DNA transfection to the cells

Here is the latest protocol for transfection of colon cancer cells:

Transfections of HCT116 using Lipofectamine 2000 and it worked perfectly in our experimental conditions.

Let me outline my brief protocol (24-well plate design);

Day 1 (Seeding):-cell seeding at a density of 100,000 cells/well (cell should be 70-80% confluent on the day of transfection)

Day 2 (Transfection):
i) Change fresh OptiMed 450 ul without PBS washing.
ii) Transfection complex
# Tube 1- 25 ul OptiMem and add 0.6 ug of plasmid DNA (firefly construct) and 0.05 ug pRL-TK (renilla control) ---then incubate 5 min at RT
# Tube 2- 25 ul OptiMem and add 1.2 ul of Lipofectamine-- then incubate 5 min at RT
# Add the content of tube 1 (diluted DNA) to tube 2 (lipofectamine) and mix gently by pipetting (2-3 times)# Further incubate 20-25 min at RT
iii) After incubation, add 50 ul dropwise directly above the cells (not to the wall of the well-plate) the tranfection complex.
iv) Incubate 16 h (Although 3-5 h is enough since transfection process is rapid, overnight incubation is OK).

Day 3 (Recovary and drug treatment):
i) Change fresh medium containing full supplement (10% FBS) and incubte 6-8 h to recovary.
ii) After recovary, wash cells once with warm HBSS and flood serum free media (I did drug treatment for 24 h in serum free condition).

Day 4 (Lysis)
i) Wash once with HBSSii) Add 100 ul passive lysis bufferiii) Freeze at -70 for 4 h or overnight

Day 5 (Luciferase)
i) Dual luciferase (Promega)Hope these help to design your own experiment.

Good luck!!

2008 Scientific meeting

When: November 7, 2008
Where: Seoul National University, Seoul, Korea

Program:
Importance of safety pharmacology for the new drug development

New drug developement from natural products

The Korean Society of Applied Pharmacology (Home Page)
Phone: 02-565-2167
Fax: 02-554-5378
E-mail: ksap1992@hanmail.net

RT-PCR protocols

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures

by
Jack Vanden Heuvel
Penn State University
Department of Veterinary Science and Molecular Toxicology Program

Click here to follow the Penn State Uni

SDS-PAGE; a nice animation by GeneEd Inc

Animation showing the process of SDS-PAGE for the separation of protein.
video

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