Week 9 Posting : Liyanah Zaffre :)

Hello my dearest friend, I hope all of you are well. It was great meeting up during the campus discussion. :)

Anyway, as for this week, I will continue from where I stop in the previous post.
After primer optimization is done, we then need to visualize the PCR products to determine at which temperature is the best. Thus, we need to load the product into the agarose gel, and subject it to gel electrophoresis.

How to make an agarose gel?
Depending on the fragment size, the percentage of the agarose gel differs. As for me, my fragment size is about 1.2kb and thus I use the 1.5% agarose gel.

Methods to make a 1.5% agarose gel
1. Select the suitable agarose gel mould and the combs, then place it in the fume hood.
2. Ensure that the mould is level, by using the air bubble gadget (air bubble must be at the centre of the gadget).
3. Calculate the amount of agarose, volume of the TAE buffer and ethidium bromide needed.

Calculation of the amount of agarose and volume of TAE buffer



For 1.5% (wt/vol) of agarose gel, we need 1.5g of agarose in 100ml of TAE buffer.
Since the suitable mould can only hold up to 60ml, we only need 60ml of TAE buffer.
100ml -> 1.5g of agarose
60ml -> 1.5/100 x 60 = 0.9g of agarose in 60 ml of TAE buffer.


Calculation of volume of ethidium bromide needed


Initial concentration of ethidium bromide: 10mg/ml
Final concentration needed for agarose gel: 0.5 ug/ml
Therefore, using M1V1=M2V2
10000ug x V1 = 0.5ug x60ml
V1= 0.5 x 60 / 10000 = 0.003ml = 3ul of ethidium bromide

4. Weigh out 0.9g of agarose powder and add it into a dry conical flask.
5. Obtain 60ml of TAE buffer using a measuring cylinder.
6. Pour the 60ml of TAE buffer into the conical flask.
7. Stuff tissue at the mouth of the conical flask and place it in the oven for 1 minute.
8. At intervals, take out the conical flask and swirl it, to check if the agarose gel has melted.
9. After which, the agarose gel solution should be clear and colourless and let the solution is cool for a few seconds.
10. Add in 3ul of ethidium bromide to the solution and swirl it again.
11. Pour the agarose gel solution to the mould and place the combs in their respective positions.
12. The gel is then left to harden and solidify for about 10-15 minutes.

Why use agarose instead of polyacrylamide?
Agarose gel is mainly used to separate smaller molecules like nucleic acids eg DNA, while polyacrylamide are capable and often used for separating larger molecules like proteins. Also, polyacrylamide gel is more expensive than that of agarose and thus, not used in this case as not necessary.

Gel electrophoresis

1. Place the agarose gel mould with the solidified agarose gel into the electrophoresis tank containing the TAE buffer. Make sure the whole gel is immersed in the buffer.
2. Add 2ul of loading dye into each of the tubes of the PCR products. Spin it down to mix it well.
3. Pipette out 10ul of the resultant mixture and load them into the respective lanes.
4. Set the electrophoresis at 120 voltage for 20 minutes, and ensure it is running.

Principle of gel electrophoresis

- Gel electrophoresis is used to separate macromolecules eg proteins or DNA that might differ in size, charge or conformation.
-When the gel is run, the molecules (eg protein in particular) will migrate towards the positive (anode) or negative (cathode) depending on their charge.
- Thus, nucleic acids foe example DNA has a negative charge due to the phosphate group attached, will migrate towards the anode.
- As noted from the methods, an electrophoresis buffer is used. Why? It is because the buffer provides ions to carry a current and also to maintain a constant pH.

Once that is done, I would then proceed on to do the gel check, to see my PCR products that will be seen as bands. For this step, a machine also known as Gel Doc is used. As this step is after primer optimization, we need to know the right temperature for the primer. The right temperature would show bright and sharp bands.

Examples



L=ladder, Temperatures: 55oC-65oC,
Best temperature: Lane 5 = 58oC

Why? Lane 1-4 shows non-specific binding. This is due to too low annealing temperatures. When that happens, the primers don’t anneal properly and thus will lead to non specific products as seen.
Lane 8-12 , although the bands are pretty bright, they are not as sharp as Lane 5.
(Sorry, pictures are not that clear so can’t really visualize the minute differences)

2nd example



As for this, there are actually 12 lanes and the ladder. However Lanes 1-6 shows smearing and non specific binding. And Lane 7 shows faint band as compared to Lane 8. Thus, it is pretty obvious in this case that Lane 8 is the best temperature. For this also, Lane 9-12 do not show any bands. This is because the temperatures are too high for the primers to anneal and thus, no products.

After which, when we have chosen the best conditions for PCR to run, we can then proceed on to do PCR for the different DNA samples at that condition. This again, will be explained in my future postings.

Alrighty, till next time then. Take care lovelies. :)

Liyanah Zaffre
0607718D
TG02

Week 8

Hey all. It's my turn to share again. For the past 2 weeks, I was attached to the Biochemistry department. Not many samples were received manually in this department as most of the samples were loaded into a sample manager and processed automatically. I believe Maya has explained what is a sample manager in one of her posts previously, so I shall not go into it. Some of the tests done in the department are urine glucose tolerance test, G6PD screening test, HbA1C test and urine drugs screening test. The rest of the tests are loaded in and processed automatically. Most of the automated tests are processed by a machine called the ADVIA 1650 by Siemens Diagnostics. However, for HbA1C test, the samples need to be ordered into the system and loaded in manually.

For now, I shall be sharing about urine drugs screening test. In the lab I am attached to, it is commonly known as Drugs 5 or Drugs 7 test. It simply means, the urine sample is being tested for the presence of either 5 drugs or 7 drugs. If Drugs 5 test is requested upon a urine sample, the 5 drugs tested for their presence are, Amphetamine (AMP), Cocaine (COC), Marijuana (THC), Methamphetamine (MET) and Opiate (OPI 300). If Drugs 7 test is requested upon a urine sample, the 7 drugs tested for their presence are Amphetamine (AMP), Cocaine (COC), Marijuana (THC), Methamphetamine (MET) and Opiate (OPI 300) with additional 2 more drugs, Barbiturates (BAR) and Benzodiazepines (BZO).
To perform these tests, special test cassettes devices manufactured by Bio-Rad Laboratories Europe Ltd. are used. It looks something like the following picture but it is not exactly the same thing. The one used in our lab tests for lesser number of drugs.

Retrived 16th August 2008 from, www.clpmag.com/issues/articles/2006-12_09.asp

For Drugs 5 test, one test device is used. The test device is able to test presence of all 5 drugs at one time. For Drugs 7 test, 3 test devices are used. 1 test device testing for the presence of the same 5 drugs, 1 test device testing for the presence of Barbiturates (BAR) and 1 more test device testing for the presence of Benzodiazepines (BZO).

The test is based on lateral flow chromatographic immunoassay to detect presence of the specific drugs and their metabolites. The principle of the test is competitive binding between either the drug present in the urine with their specific antibody or, the drug conjugate with the specific antibody.

3-5 drops of urine are dropped onto the each of the circular wells found at the bottom part of the test device. The urine will then move upwards by capillary action. If the drug is present below the cut-off concentration, the drug conjugate will bind to the specific antibody instead and forms a visible line across. If a visible line is observed, the result is recorded as negative. However, if the drug is present in high amounts and above the cut-off concentration, the drug will bind to the specific antibody instead of the drug conjugate. This will cause no visible line to be observed across and the result is recorded as positive. For every drug tested, there is a region for Control. If there is no visible line observed across at the Control region, the test is then considered invalid and should be tested again using a new test device.

The following figure is a visual interpretation of the results, done using Microsoft Powerpoint.

The cut-off concentrations are as follows for each type of drug:-

Amphetamine - 1000 ng/mL
Barbiturates - 300 ng/mL
Benzodiazepines - 300 ng/mL
Cocaine - 300 ng/mL
Marijuana - 50 ng/mL
Methamphetamine - 1000 ng/mL
Opiate (OPI 300) - 300 ng/mL

The antibodies present on the test devices for each drug type are mouse monoclonal antibody-coupled particles. For each drug test, drug-protein conjugates are also present specific to the drug type. For each Control region, it contains goat anti-rabbit IgG polyclonal antibodies and rabbit IgG.

This urine drugs screening test is only a qualitative test and not a quantitatinve test. Therefore, we are unable to tell the amount or drugs level present. It is also only a screening test. A gas chromatography or mass spectrometry should be carried out to confirm any positive results obtained. A negative result does not necessarily the urine is free of drugs. Other types of drugs not tested for their presence, may also be present. A positive result may also be caused by the kind of food consumed by the patient.

As most of the drugs are controlled drugs, I have never encounter any positive results ever since I have been attached to the Biochemistry department. According to one of the permanent staffs there, it is also quite rare to encounter any postive results as Singapore rules are very tight when it comes to abusing drugs.

I guess that's all for now. If you have any queries to ask, please feel free to do so. I will try to reply as soon as I can. Thank you. C:

Nur Azeimah
0607060A
TG02