Science

RE: [DIYbio] DIY Seattle Lab

5:29 PM |

Hi Jacob,

I live in Seattle and have been interested in getting a lab going for quite some time. I currently have many bits and pieces, but haven’t put it all together yet. Right now, most of it is in storage while I look for a suitable place to set up shop. I would be particularly interested in your microscopes, but it sounds like you have a lot of great equipment. Perhaps we can chat a bit on the phone. My cell is 425-503-5862.

-Kris

From: diybio@googlegroups.com [mailto:diybio@googlegroups.com] On Behalf Of Jacob Avera
Sent: Saturday, June 30, 2012 1:22 PM
To: diybio@googlegroups.com
Subject: [DIYbio] DIY Seattle Lab

HI I've got thousands of dollars in lab equipment I've built up. I would like to give it to someone in Seattle. I need to go take care of a relative. Mostly older Lab equipment but top of the line when new. Spec 20 Reflectometer, Fisher 13k fuge, stuff for PCR .
Gilson pipettors etc. plus a trunk(size) of glassware, a water distiller (needs a part from hardware store) ETC......

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Re: [DIYbio] Where to buy PCR primers/oligonucleotides?

5:16 PM |

On 06/30/2012 01:35 PM, jarlemag wrote:
> I've looked up quite a few suppliers, but all of them require listing which organization you belong to for registering and/or
> shipping.

So, you create a company identity for yourself. File a doing business as fictitious name in your county.
Pay for the order with a check from the dba account with the fictitious name. That's all you need... well,
bookkeeping and a few other things maybe...and taxation, of course.

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[DIYbio] SNPs (genetic markers) for exceptional longevity in humans - data

3:34 PM |

Supplementary data from publication which includes the SNPs (see table S1) below.
Who wants to make a test kit?

http://www.sciencemag.org/content/suppl/2010/06/30/science.1190532.DC1/Sebastiani_SOM.pdf

Video, http://www.youtube.com/watch?v=uPE7S27O5ok

Publication abstract (note there is a retraction to this paper which qualifies and lessens the 77% accuracy result, due to bugs in their protocol & equipment):

"""ABSTRACT

Healthy aging is thought to reflect the combined influence of environmental factors (lifestyle choices) and genetic factors. To explore the genetic contribution, we undertook a genome-wide association study of exceptional longevity (EL) in 1055 centenarians and 1267 controls. Using these data, we built a genetic model that includes 150 single-nucleotide polymorphisms (SNPs) and found that it could predict EL with 77% accuracy in an independent set of centenarians and controls. Further in silico analysis revealed that 90% of centenarians can be grouped into 19 clusters characterized by different combinations of SNP genotypes—or genetic signatures—of varying predictive value. The different signatures, which attest to the genetic complexity of EL, correlated with differences in the prevalence and age of onset of age-associated diseases (e.g., dementia, hypertension, and cardiovascular disease) and may help dissect this complex phenotype into subphenotypes of healthy aging."""

References:

* Discussion of retraction, http://uncertaintist.files.wordpress.com/2011/08/science-retraction-2011.pdf

* Supporting Online Material for Genetic Signatures of Exceptional Longevity in Humans, http://www.sciencemag.org/content/suppl/2010/06/30/science.1190532.DC1/Sebastiani_SOM.pdf

* Genetic Signatures of Exceptional Longevity in Humans, http://www.bumc.bu.edu/medicine/files/2010/07/longevity-genes.pdf DOI: 10.1126/science.1190532

* Youtube interview with researchers, http://www.youtube.com/watch?v=uPE7S27O5ok

## Jonathan Cline
## jcline@ieee.org
## Mobile: +1-805-617-0223
########################

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Re: [DIYbio] Favorite Tools for Aligning Multiple Sequences

2:22 PM |

Alright, so you're doing multiple sequence alignments? Like, global alignments?

Finally, a chance for me to be useful. I don't do synthetic biology, but I am interested in evolution, and, unsurprisingly, multiple alignment is very, very important to molecular phylogenetics-- if your alignment is no good, your phylogenetic reconstruction will be no good. However, picking an alignment method isn't that easy; "The Phylogenetic Handbook" lists, IIRC, some 43 different alignment algorithms. They take fundamentally different approaches, too; my favorite, which seems somehow thematically appropriate, is that of genetic algorithms.

"The Phylogenetics Handbook" has this to say about Clustal:

> Although ClustalW is still the most popular alignment tool, several more recent methods have now been shown to perform better in terms of accuracy and/or speed.

It has a nice table under the "Which program to choose?" section. You're probably in the realm of the first case:

> Input data: 2-100 sequences of typical protein length (maximum 10,000 residues) that are approximately globally alignable
>
> Recommendations: Use ProbCons, T-Coffee, and MAFT or MUSCLE, compare the results using AltAVisT. Regions of alignment are more likely to be correct. For sequences with low percent identity, ProbCons is generally the most accurate, but incorporating structure information (where available) via 3DCoffee (a variant of T-Coffee) can be extremely helpful.

Personally, I tend to use T-Coffee. The tradeoff here is between accuracy and computational requirements: T-Coffee is generally more accurate, but Clustal has lower memory requirements. I think Clustal is currently in the realm of "everyone uses it because everyone else uses it".

I'm no expert, either, so take this with the requisite grain o' salt.

--T.

On Jun 25, 2012, at 5:27 PM, L wrote:

> Hey there everyone,
> I'm looking for a way to quickly create protocols for transformation of artificial yeast chromosomes. Got lots of ideas, the means to try them all, and now I'm just looking into what restriction enzymes I'll need. Confused by BLAST, any help using that would be awesome.
>
> Thank you!
>
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Re: [DIYbio] Translating bacterial DNA into plant DNA

2:04 PM |

Hey Mega,

Nice post! I've actually been thinking a lot about this as well!

Not sure if you saw but there was a group that already put the lux operon in plant chloroplasts:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015461

Right now, I'm working to get some tools rounded up and protocols for Arabidopsis transformation working. But one thought I had about cloning out the vibrio lux operon was using the viral 2a peptide sequence (there are several articles about it but here is one: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018556)

While I don't have any experience with the 2a peptide sequence, in theory we could express two (or three!) genes separated by the sequence for the 2a peptide from the same promoter and during translation the 2a peptide sequence would result in two protein products. So rather than having to transform a plant with the 5 lux genes with 5 promoters etc. we could transform the plant with two or three 'genes'.

If you're interested, I'd be up for collaborating with you!

Thanks,

Kyle

On Monday, June 11, 2012 11:52:35 AM UTC-7, Mega wrote:

I'll be doing it the coward methode.
Promotor-RBS-LuxA-Terminator -- Promotor-RBS-LuxB-Terminator ..... C-D-E

The most expensive it would be that it doesn't work. And one'd have to order it again...

In the database I found all the lux genes. The gene sequnece does not contain promoter nor RBS or terminator if it's just called "LuxA from Vibrio Fischeri strainxxxx"???? Because it does say only "gene" but not "promoter" . So I'm free to just take that seqeunce and attach a good promoter?

2012/6/11 Cathal Garvey <cathalgarvey@gmail.com>
Actually, generally Eukaryotes need one-promoter-per-CDS (CDS = "Coding
Sequence). So for each protein coded by the operon, you'll need to
extract the coding sequence, and provide it with its own
promoter/rbs/cds/terminator setup.

Yes, this is unfortunately very costly! It's due to the difference in
how bacteria and eukaryotes start reading mRNA: prokaryotic ribosomes
*assemble* on the RBS, whereas eukaryotic ribosomes first bind the
modified-G at the 5' end of the mRNA, and then travel downstream until
they find an RBS, whereupon they start translating. But, when they hit a
stop codon, they dissociate, so additional CDS's get ignored.

There are so-called "Internal Ribosomal Entry Sites" for Eukaryotes,
usually from viruses. I don't know if there are any for plants, or for
your target species of plant. They are often virus-derived, and may act
as a warning signal to plant innate defence systems. I simply don't
know. If you do find any "IRES" for your plant species in the
literature, it would save you a lot of extra DNA, because you could then
re-design the operon to work as an operon in plants, instead of needing
~8 distinct genes!

Consider however: A lot of the genes in the Lux operon code for proteins
that often work in tight association together. For example, LuxAB form
the actual luciferase, which does the job of digesting mature luciferin
to create light. LuxCDE work to create mature luficerin. I think that
LuxFGH are regulatory genes, LuxI certainly is: You mightn't need these
if you replace them with a plant regulatory signal instead.

If that leaves only LuxAB and LuxCDE, it's possible you can create
"Chimeric Proteins" of these two sets that will still work. Sometimes,
protein chimeras work at a higher efficiency, because the product of one
protein in the chain is ejected in close proximity to the active site of
the next protein in the chain.

To make protein chimeras, simply fuse the CDSs, removing the STOP codon
of the first protein and replacing it with a "spacer stretch" of fairly
boring/neutral, hydrophilic amino acids. Then surround this hybrid CDS
with a Promoter/RBS + Terminator as if it were only one protein.

If all goes well, the two sub-proteins will fold correctly, joined by a
"boring" spacer of inert amino acids, and will work as intended. The
benefit: You've saved a lot of promoter/Terminator DNA. It's even
possible that metabolic chains such as LuxCDE will work even better as
fusions.

On the other hand, the connection of three distinct proteins, each with
their own methods of self-folding, may result in a protein folding
train-wreck which utterly fails to work. In which case, far from saving
money spent on DNA, you've lost all the money spent on your protein
fusion. It's a gamble!

On 09/06/12 17:05, Mega wrote:
> Thought it would be best to start a new thread for this.
>
> I had the light producing genes (Lux ABCDE) rewritten by a program to adapt

> it to *Arabidopsis *(Codon Bias).

>
>
> Now I have to add promoter(s), RBS and terminator.
>
> According to this
> http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pJE202 , in the native
> plasmid there is only one promoter "Vibrio fischeri luxICDABEG operon
> promoter".
> Translating this into plant genes, I just need one promotor for all the
> genes?? (Virus-Plant promoter, because of strong expression)
>
> When I attach the promoter sequence to the gene segence, does there have to
> be sapce left or just add it like ATGTC + AAAA = ATGTCAAAA ??
>
>
> I think this would be the best promoter available: CLCuMV C1. Does anyone
> know where to get the sequence from??
>
>
> thx
>

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[DIYbio] DIY Seattle Lab

1:22 PM |

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Re: [DIYbio] Re: BiohaX Team - Help Wanted!

12:18 PM |

i don't quite understand your question

planning here and on Initiatr hasn't been going great so i thought a live group conversation would be good to start brainstorming, it doesn't have to be G+

On Saturday, June 30, 2012 12:04:21 PM UTC-7, Eugen Leitl wrote:

On Sat, Jun 30, 2012 at 01:03:36AM -0700, QDragon Leet wrote:
> we're kinda stalling here
> what do you think about a G+ hangout to discuss the series?

Would you create a particular account with a particular social network
you took great pains to stay away from just to be able to follow a particular
discussion?

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Re: [DIYbio] Re: BiohaX Team - Help Wanted!

12:04 PM |

On Sat, Jun 30, 2012 at 01:03:36AM -0700, QDragon Leet wrote:
> we're kinda stalling here
> what do you think about a G+ hangout to discuss the series?

Would you create a particular account with a particular social network
you took great pains to stay away from just to be able to follow a particular
discussion?

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Re: [DIYbio] Re: Genetic tests to do on foodstuffs to test PCR

11:44 AM |

1. That ladder suggests that you need to run the samples longer. It's not separating effectively. The gel size and the amplitude of the current also affect how much 'oomph' you need to pull the bands through the gel. Consider extending this time, if possible. This will also help you separate two sized bands in males (XY).

2. yes to the other comment on etoh extractions. phenol will kill your pcr.

3. where are you getting the reagents for the PCR reaction? if its a kit (or even ask for a free sample from some companies) follow their instructions if you aren't already. I've found them to be more robust in their guidelines than old papers (i assume its old since it lists the dNTP concentrations, and I don't see that anymore in recent papers).

For the Sigma kits that are linked on the wiki, these are hot-starts, and require something like a 5min denaturation to remove the antibody (or whatever it is in that kit) that prevents non-specific products from forming before the reaction 'should' begin. it's a great feature, but if you don't account for that in your pcr reaction, you'll get ambiguous bands... and none of the gels really convince me you/they have a product yet (on that wiki).

if my suggestions dont make sense, just let me know.

On Sat, Jun 30, 2012 at 1:30 PM, TRolandB <williambeaufoy@gmail.com> wrote:

Thanks to all for the advice. In reply

- The ladder always works, so no problem with the electrophoresis I would think.

- The fragment sizes are 977 and 788bp, quite large fragments I think?
- Good idea on the multiple PCRs, we may give that a go.
- on the page I link to there's a section detailing our extraction protocol: complete protocol as of June 2012. - we use chelex.

On Friday, 29 June 2012 01:35:16 UTC+1, cory....@gmail.com wrote:
The first step would be to really figure out which step is the
problem. Have you tried doing multiple PCRs on the same DNA
extraction?

My suggestion would be to do 3 different DNA extractions all from the
same person. Then do 3 different PCR reaction on each sample, mixing
the PCR reagents separately for each reaction, then (assuming you are
confident that your thermalcycler is consistent across wells) run all
the reactions at the same time and then load them all into the same
gel side by side. From there you should be able to tell if there is
variability between extractions, between PCRs, or both.

Also, what exact protocol are you using for the extraction? Your wiki
links to a couple of different protocols. If you're using
phenol/chloroform I would suggest doing multiple ethanol
precipitations as phenol inhibits PCR.

-cory

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[DIYbio] Where to buy PCR primers/oligonucleotides?

11:35 AM |

Hi. So, I just got my OpenPCR kit, which I must admit I bought somewhat on a whim. I'm a Msc. student in biotechnology, and like the idea of doing some "research" on my spare time. I'm participating in our university's iGEM team, and for the last couple of weeks we've been working full-time on our project and getting more lab experience. We haven't gotten to running PCR yet, but that's coming up. It would be fun to put my own machine to use too (if I can manage to assemble it). I'm having one problem though - where can I get primers synthesized and the other reagents I need (I don't want to take anything from the lab, and would like to be able to design and buy my own primers separate from our project)?

I've looked up quite a few suppliers, but all of them require listing which organization you belong to for registering and/or shipping.

The suppliers I have checked out include:

eurofins mwg|operon
IDT DNA
Invitrogen/Life Technologies
Sigma Aldrich
VWR
GenScript
BioLegio

Does anyone know of any suppliers that ship to residential addresses, and don't require an institutional affiliation?

Thanks for your attention,

Jarle

PS: I'm located in northern Europe

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Re: [DIYbio] Re: Genetic tests to do on foodstuffs to test PCR

11:30 AM |

Thanks to all for the advice. In reply

- The ladder always works, so no problem with the electrophoresis I would think.

- The fragment sizes are 977 and 788bp, quite large fragments I think?
- Good idea on the multiple PCRs, we may give that a go.

- on the page I link to there's a section detailing our extraction protocol: complete protocol as of June 2012. - we use chelex.

On Friday, 29 June 2012 01:35:16 UTC+1, cory....@gmail.com wrote:

The first step would be to really figure out which step is the
problem. Have you tried doing multiple PCRs on the same DNA
extraction?

My suggestion would be to do 3 different DNA extractions all from the
same person. Then do 3 different PCR reaction on each sample, mixing
the PCR reagents separately for each reaction, then (assuming you are
confident that your thermalcycler is consistent across wells) run all
the reactions at the same time and then load them all into the same
gel side by side. From there you should be able to tell if there is
variability between extractions, between PCRs, or both.

Also, what exact protocol are you using for the extraction? Your wiki
links to a couple of different protocols. If you're using
phenol/chloroform I would suggest doing multiple ethanol
precipitations as phenol inhibits PCR.

-cory

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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel CS604- Operating System Assignment No 5 Solution by VIRTUALIANS.NING.COM

10:06 AM |

Kindly click on following link and see assignment solution.

http://virtualians.ning.com/group/cs604-assignments/forum/topics/assignment-no-05-semester-spring-2012-cs604-operating-system

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There is a great chat room you can easily chat with VU fellows and others for study discussion and also share your views here on VIRTUALIANS.NING.COM.

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Sat, 30 Jun 2012 - Today Jobs on all Leading Newspapers of Pakistan

5:20 AM |

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[DIYbio] Free plasmids for amateur biologists.

4:03 AM |

Hello! I have found the way to get some free plasmids with map and work protocol. The Aldevron company distributes free samples of pRc/CMV-Hbs(S) and pCMVHB-S2.S plasmids. Both are based on pBlueScript plasmid and contain Ampicilline resistance gene. Each sample contains 2-4 ug of plasmid DNA. Samples are delivered via postage service. Link to order samples: http://www.aldevron.com/products/dnas/plasmids/

I hope it'll be useful for somebody.

P.S.: sorry for my bad English.)

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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO403 - Final TERM Paper Solved/Unsolved Materials

3:49 AM |

ECO403 - Final TERM Paper Solved/Unsolved Materials

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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO402 Final TERM Paper Solved/ Unsolved Materials

3:20 AM |

ECO402 Final TERM Paper Solved/ Unsolved Materials

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Re: [DIYbio] Odd gel run following PCR

3:00 AM |

I agree primer-dimer at the bottom of the gel. Bad design isn't the only reason to see them though. They are often seen with lack of template. So even though dimer formation is not favourable in the abscence of anything else to bind to the stick to themselves.

Zeb

Sent from Samsung Mobile

Cathal Garvey <cathalgarvey@gmail.com> wrote:
Those fuzzy bands at the bottom, if they are not loading dye, are 'primer dimers'. You get them when your primers are able to prime one another or self-prime, causing lots of wasted primer and 'distracting' the the reaction from the real product.

One way to avoid this is to carefully avoid primers which can prime one another. Another way is to raise the annealing temperature step in your reaction to somewhere closer to the primer melting temperature, so that it's less likely that only-partially matching DNA will bond.

pcrbro <princeofnam@gmail.com> wrote:

>
>
>The following is my 1% PCR gel run.
>
>As you can see the products have strangely run all the way until the
>end. The products also look to be further than the dye looks to be on
>the
>gel itself. I'm relatively new to PCR and not completely sure how to
>trouble shoot this. The first row is a 1 kb ladder and the third row
>is a
>control. The expected product should have had a product length of 611.
> I
>ran the PCR at 4 annealing temperatures 5 C below the Tm. Does anyone
>know
>where I should start in determining what may have gone wrong with my
>PCR/gel run?
>
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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel MGT602 Answer Required

1:06 AM |

I need the correct Answer of the following Question, anyone please provide

A marketing Plan answers the _______ types of questions

Select correct option:

Which one of the following plans should contain control points to ascertain progress and to initiate contingency plans if necessary?

Financial plan

Marketing plan

Business plan

Operational plan

Which of the following section of business plan provides complete overview of the product, service and operations of a new venture?

Production plan

Marketing plan

Description of venture

Industry analysis

Which of the following business size has lot of adjustment capacity against the changes?

Small

Middle

Large

High growth

Which one of the following contracts should be in written?

Real estates

Lease

Rentals

All of the given options

The members of the board should be carefully selected by considering which of the following criteria?

Select individuals who can work with a diverse group and will commit to the venture's mission

Select candidates who understand the market environment

Select candidates who will show good judgment in business decision making

All of the given options

"Decide what general market or industry you wish to pursue" is the first step of:

Segmentation

Saturation

Marketing plan

Marketing system

Which of the following term describes the situation of two or more firms that offer essentially the same good or service in the market?

Direct channel

Indirect competition

Direct competition

Indirect exports

Job description should include all of the following EXCEPT:

The major duties relevant to a particular job

Expected performance standards

The equipment needed by the worker

An evaluation of the worker's personal needs

Which of the following business form has advantage of no capital stock tax or penalty for retained earnings in the business?

Proprietorship

Limited liability company

Corporation

S corporation

A short-term, internal source of funds can be obtained by reducing all of following EXCEPT

Short-term assets

Cash

Fixed assets

Inventory

Which form of the business has the most freedom in terms of selling one's interest in the business?

Sole proprietorship

Partnership

Corporation

S Corporation

SBA provides:

Finance without interest

Finance with interest

Guarantees for getting loans

Financing up to 90 % of principal amount

In USA Public Limited Companies may have ______ Director/s

On the part of entrepreneur which of the following has/ have a great concern to the Investors

Experience

Commitment

Knowledge

All of the above

On which of the following factors the death or withdrawal of a limited partner has no effect?

Continuity of the business

Cost of the business

Ownership of the business

Growth of the business

Before leaving his job as a brand manager for a leading toy manufacturer in France, John smith was asked to update his __________,including the job's objectives, type of work he did and other responsibilities.

Job analysis

Job description

Job specification

Job evaluation

SBIR grant program is controlled by:

10 Agencies

11 Agencies

12 Agencies

13 Agencies

Equipment can be used to secure long term financing, usually on a ______ basis.

1 to 2 years

3 to 6 years

3 to 10 years

4 to 5 years

A marketing Plan answers the _______ types of questions

Targeting the market consists of ______ Procedures

Corporations generally possess not more than two of the following four characteristics: (1) limited liability, (2) centralized management, (3) one time taxation, and (4) least expensive to start. The two characteristics most likely to be absent in corporations are __________.

Limited liability and centralized management

Centralized management and one time taxation

Centralized management and least expensive to start

One time taxation and least expensive to start

Before setting the product price, which of the following elements the entrepreneur needs to consider?

Costs

Markups

Competition

All of the above

From which of the following R & D limited partnerships provide funds to entrepreneurs in high technology fields?

Equity contribution from investors

Debt from lenders

Cash from company operations

Investors looking for tax shelters

Which of the following is the most important for the entrepreneur, while starting a new venture, to make an assessment of?

Risk

Profit

Market

Competitors

Which of the following is used to finance floor plans of retailers such as automobile and appliance dealers?

Line of credit

Trust receipt

Accounts receivable

Real estate

Which of the following is NOT an example of gathering data from secondary sources?

Trade magazines

Newspaper

Government agencies

Focus group

Which of the following financing does not require any collateral and offers the investor some form of ownership position in the venture?

Commercial bank loan

Line of credit

Equity

Character loan

Corporation has______ Taxation system

Triple

Single

Double

Which of the following section of business plan provides complete overview of the product, service and operations of a new venture?

Production plan

Marketing plan

Description of venture

Industry analysis

Which of the following statement is calculated from projected revenues minus projected costs?

Pro forma income

Pro forma expense

Pro forma revenue

Pro forma sales

Which of the following is a major disadvantage of the corporate form of business?

Double taxation of dividends

Limited liability of shareholders

Transferability of interest

Inability of the firm to raise large sums of additional capital

In which of the following royalty partnership and joint venture are used to reap the benefits of the effort?

Funding stage

Development stage

Growth stage

Exit stage

Which one of the following best describes the flow of goods and services from production to the customer?

Organizational plan

Marketing plan

Operations plan

Financial plan

Which of the following is included in the description of the business?

Location

Products offered

History

All of the given options

Which statement of the following relates with the ultimate goal of Business

Profit Statement

Goal Statement

Mission Statement

Vision Statement

Which of the following factors can be directly influenced or controlled?

Economy

Material price

Inflation

Fuel price

Banks are usually reluctant to give loan to the

Private Limited Companies

Partnership firm

Public Limited Companies

Sole Proprietor

Investors usually prefer the income statement which projects the sales volume of _____ years

Which one of the following responds to the question of: "Where do we want to go?" and specify things such as market share, profits, sales, market penetration etc.?

Marketing goals and objectives

Mission statement

Vision statement

Market strategy and action programs

Which variable of the following has the critical decisions of media alternatives, message, role of personal selling, media budget and coupons, etc?

Product

Price

Channel of Distribution

Promotion

What is the primary concern of founders who trade equity for capital for their growing

venture?

Select correct option:

Capitalization

Investor capabilities

Valuation

Control

All of the following are the major components of any research and development partnership EXCEP

Contract

Sponsoring company

Tax shelter

Limited partnership

Which statement of the following relates with the ultimate goal of Business

Select correct option:

Profit Statement

Goal Statement

Mission Statement

Vision Statement

LLC is more flexible than

Select correct option:

S Corporation

Limited Partnership

Sole Proprietor

All of the above

On the part of entrepreneur which of the following has/ have a great concern to the

Investors

Select correct option:

Experience

Commitment

Knowledge

All of the above

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

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[DIYbio] Re: BiohaX Team - Help Wanted!

1:03 AM |

we're kinda stalling here

what do you think about a G+ hangout to discuss the series?

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Re: [DIYbio] Google+ Hangouts for DIYbio?

1:01 AM |

i'd also be very interested in something like this

is there a G+ group?

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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel Re: ECO401 - FInal TERM Paper Solved/ Unsolved Materials

12:36 AM |

On Sat, Jun 30, 2012 at 12:32 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:28 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:26 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:22 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

ECO401 - FInal TERM Paper Solved/ Unsolved Materials

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

Read User's Comments(0)

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO401 - FInal TERM Paper Solved/ Unsolved Materials

12:32 AM |

On Sat, Jun 30, 2012 at 12:28 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:26 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:22 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

ECO401 - FInal TERM Paper Solved/ Unsolved Materials

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

Read User's Comments(0)

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO401 - FInal TERM Paper Solved/ Unsolved Materials

12:29 AM |

On Sat, Jun 30, 2012 at 12:26 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

On Sat, Jun 30, 2012 at 12:22 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

ECO401 - FInal TERM Paper Solved/ Unsolved Materials

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

Read User's Comments(0)

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel Re: ECO401 - FInal TERM Paper Solved/ Unsolved Materials

12:26 AM |

On Sat, Jun 30, 2012 at 12:22 PM, Umair Saulat <saulat.umair@googlemail.com> wrote:

ECO401 - FInal TERM Paper Solved/ Unsolved Materials

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

--
Zindagi mein 2 Logo ka buhat khayal rahkoooo

Ist woh jiss ney tumhari jeet ke Liye buhat kuch hara hoo (Father)

2nd woh jiss ko tum ney har dukh me pukaara hoo (Mother)

Regards,

Umair Saulat

Read User's Comments(0)

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[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel Re: ECO401 - FInal TERM Paper Solved/ Unsolved Materials

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO401 - FInal TERM Paper Solved/ Unsolved Materials

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel ECO401 - FInal TERM Paper Solved/ Unsolved Materials

[Virtual University Of PakistanStudent Cafe] .::VULMSIT::.eNoxel Re: ECO401 - FInal TERM Paper Solved/ Unsolved Materials