It is the 14th week!!!…..left 6 more weeks to go. Jiayou pple. As I am still in a chemistry lab for the last 14weeks and for the future 6 weeks, this week post will continue from my first post. (week 4) which is on my major project which is on phytoplankton (diatom and dinoflagellate are some e.g. of phytoplankton.)
Here is a recap:
1) Seed phytoplankton cells
2) Harvest (centrifuge) when culture is ready
(Centrifuging takes 1 whole day. Why? Cause got about 20 litre of culture to spin down and there is only 1 rotar available----1 rotar can only carry 6 bottles, each bottle has a volume of 250ml, so after calculating…figure out !?!)
3) On the supernatant, carry out SPE
a. Methanol loaded first to condition the cartridges and wash away any impurities
b. Then load DI water to remove methanol
c. Next, load the supernatant (this take many days)
d. Then, load DI water again to remove salts (cause seawater contains salts)
e. Spin down the cartridges to ensure all DI water is removed from cartridges
f. Lastly, load methanol to elute out the extract.
So, now I have the eluent which contains the solvent (methanol) with the extract I want.
To get the extract and remove the methanol, evaporation is carried out.
Technique: evaporation
Used for removing solvent
Equipment used: rotary evaporator
(Image taken from http://www.eyelausa.com/category.php?cID=1)
It is a very easy process but take very very long for large quantity of eluent.
The bottle on the right is the evaporating flask and on the left is the receiving flask.
Water aspirator is connected to the hook-like thing on the condenser. Condenser pump is placed in a beaker with water covering the pump and the remaining space in the beaker is filled with ice.
So first switch on the condenser (the tall tube with coil inside) and water bath (it take time for the water bath to reach the temp set).
What temp to set for the water bath?
Temp set is around 40Cto 50C as long as it is below methanol boiling point.
Depending on the compound want to obtain, if it is stable and does not react easily (meaning it has few hydroxyl(OH) groups), then it is alright to set at higher temperature but the temp must be below the boiling point.
Eluent is poured into the evaporating flask, filling around 1/3 of the flask. If the flask is filled too full, eluent might spill out while evaporating and the whole process need to be repeated again.
After everything is set, the water aspirator is on. Water aspirator provides the vacuum required. Next, the pressure is gradually reduced. The eluent in the evaporating flask had to be monitored constantly in case there is lots of bubbles (‘boiling”). When this occurs, the pressure is released a while then reduced again. If violent boiling occurs, the desired compound may spill over to the receiving flask.
When evaporating, methanol from the evaporator become vapours and travels up to the condenser. The vapours then condense on the cool surface of the condenser. The methanol in liquid form then dripped into the receiving flask.
This whole process continue until the eluent in the receiving flask left a very small quantity, then I top it up with more eluent. When all eluent has been poured into the evaporating flask, then I evaporate the remaining eluent till dryness.
And the residue in the evaporating flask is the extract that I want to get. The residue is of a very very small quantity. To get sufficient amount for LCMS (discuss about it in next post) …. I need to accumulate about 3 times of SPE eluent.
After accumulating, I use methanol to dissolve and suspend the accumulated dry extract. Then I transfer the extract into a small tube using a dropper. As there may be some extract then cannot be removed from the walls of the flask, sonication using a sonicator is carried out to detach the extract from the walls.
Next I dry the extract in the tube using stream of nitrogen gas. (Why nitrogen? Actually nitrogen is a common gas used for drying.)
For the cells,
Methanol is used to remove the cells from the centrifuge bottles.
The methanol containing the cells is transferred into a conical flask.
The cells is left in the flask for about 1 week. Meanwhile, the flask is sonicated.
Methanol can lyse the cells to get the extract from the cells.
Sonication helps to enhance the process of cell lysis because high frequency sound waves can shear cells.
After that, the mixture (cells in MeOH) is filtered to remove the cells.
Methanol extract is evaporated as mentioned above.
LCMS on the extract will be discussed in the next post.
6 more weeks to go.
Hope my explanation is clear.
Any doubts, questions or uncertainty, juz ASK. =D
Lim Xin Ni
TG02
0607325H
Department: Biochemistry
Topic: Processing of specimens and LX PRO Analyser
Hey all, i'm in biochemistry lab now.
I'm going to talk about the processing of biochem samples and about the LX PRO Analyser.
Firstly when samples are reeceived in the biochem lab, the med tech has to check a few importatnt things.
1. Patient's name
2. Patient's IC no
3. Accession no
4.Tests ordered and collection priority: Urgent or routine
Samples are being spinned down as the serum is the one tested by the Analyser.
Samples that are in the primary tube could be directly placed on the rack with the adaptor. While the samples in the microtube should be proceed differently. The serum should be pipetted into the sample cup and a LIS acession label should be put on the sample cup to allow the analyser to read it.It must be checked that there are no bubbles in the sample cup as it will affect the results.Then only should it be placed on the rack with the adaptor.Rack is then placed on the autoloader of the Beckman LX PRO analyser.
Beckman LX PRO analyser is a machine that conducts all kinds biochemical tests.
It includes urea, glucose, creatinine, albumin, uric acid, total protein, Phospurous, magnesium, gentamycin etc.
Before patients' samples could be run, QC have to be done every morning.
The level of the modular chemistry agents should be checked. The ones with low levels should be loaded.
Next calibration has to be done.The analyser analyzer performs the calibrations. It automatically checks the calibration and produces the data at the end of the calibration. Error codes will be printed out against failed calibrations. Troubleshooting has to be done against failed calibrations and only if calibration passes,we can proceed to run controls.
QC schedule plan has the chemistries to do the QC run. Controls should be mixed well and then dispensed at the required volume into the specified control into the sample cup. Analyzer runs all the chemistries for that control. Troubleshooting has to be done for flagged chemistries. Control check has to pass before proceeding to run patient samples.
That's it.It's not very difficult, pretty easy.
Cya all:D
Neela
TG02
For this week, we are supposed to post an entry on any test that we have done. Therefore, I will share with all of you a test to detect for G6PD deficiency.
Introduction
G6PD (Glucose-6 Phosphate dehydrogenase) is a key enzyme in needed for the formation of NADPH in the hexose monophosphate pathway. HADPH is important for maintaining the integrity of the RBC(erythrocyte) membrane and functioning properly.
G6P-DH
Glucose-6-P + NADP+ <======> Gluconate-6-P + NADPH + H+
The lack of G6PD is the cause of hemolytic disease in newborn. It is an inherited condition where the body does not have enough G6PD. It can be also be transmitted as X-linked recessive. It can cause hemolytic anemia, usually after exposure to certain drugs (e.g. some oxidant drug) or food etc.
Principle of test
The name of this test is called the Fluorescent spot test. It is basically mixing the working substrate with the blood sample. After that, it is pipetted onto a filter paper. If NADPH is produced in the reaction, when the filter paper is placed under long wave UV light, the spot of blood will fluorescence.
Methods
1) G6PD working stock is removed from the refrigerator
It contains
- 1 tube of positive control
- 1 bottle of working substrate
2) One micro tube of intermediate control and one deficient control is taken out from a
-20degree Celsius freezer and then thawed.
3) For every specimen, checked that the name and the IC number on the form and the specimen bottle tallies. Also, check that the test ordered is correct as indicated on the sticky label.
4) The tubes were to be prepared for loading of specimen according to the number of specimen and label accordingly. Also, label 4 other tubes; blank, intermediate, deficient and positive
5) 100ul of substrate was pipetted into each tube at room temperature.
6) 5ul of the patient’s specimen is added into the specific tubes. Also, 5ul of intermediate, deficient and positive is allocated into the specific tubes. The mixture is pipetted up and down several times to mix it.
7) Allow 10minutues for the reaction to take place. Time is taken using a timer.
8) The filter paper that is provided in the kit is to be labelled with the number
9) After 10minutes is up, 10ul of the mixture from each tube of the patient’s samples are added to the filter paper. Also, 10ul of the blank, intermediate, deficient and positive are also added to the filter paper.
10) The filter paper is then placed into the incubator to allow it to dry completely (approximately 15mins)
11) After 15mins, take out the filter paper and view it under UV light.
12) Patient’s sample with normal G6PD should show strong fluorescence while patients. Samples that fail to fluoresce may indicate marked deficiency or total lack of G6PD enzyme.
13) It is reported as positive for samples that fluoresces and test is repeated for weak or no fluorescence.
For the specimen that test is needed to be repeated, check the specimen tube to see if there are any clots present, if there are no clots, then the test repeated over again to confirm. If there are clots, then inform the ward for a repeat of test with a new specimen.
That's about all =)
Zhenling
TG02
Erm, Hi. haha. Anyway, I decided to talk about HBeAg that is done in the lab using Architect machine at the Immunology sector.
HBeAg determination is used to monitor the progress of hepatitis B viral infection.
Hepatitis B viral infection could be contracted via contact with infectious blood, body fluids from having sex with an infected person, sharing contaminated needles to inject drugs/ reused during treatment, or from an infected mother to her newborn.
HBeAg can be detected in the early phase of Hep B infection after the appearance of Hep B surface Ag. Both increases significantly during the viral replication in acute infection. HBeAg's presence correlates with increased no. of the infecious virus aka Dane particles, the occurence of core particles in the nucleus of the hepatocytes (liver cells, presence of hep B viral specific DNA and DNA polymerase in the patient's serum.
The virus reproduces transporting into the liver by first attaching to the membrane. The cpre particle then releases its DNA and DNA polymerase contents into the liver cell nucleus. Once it enters within the cell nucleus, the Hep B DNA causes the liver cell to replicate the DNA and produce more copies of the viral DNA. Then it produces copies of the virus within the cell before being released into the blood stream.
Architect uses Chemiluminescent microparticle Immunoassay (CMIA) technology and the presence/absence of HBeAg is determined by comparing the chemiluminescent signal in the reaction to the cutoff(CO) signal. If it is lesser than the CO signal, the sample is considered to be nonreactive for HBeAg.
Usually blood collected from plain tubes are used and the interpretation of the results are as follow:
Specimen with S/CO values < 1.000 = Non-reactive
Specimen with S/CO values >or= 1.000 = Reactive
Yup, thats about it for now. I'm open to questions, but pls don't make things too hard for me. Haha.
Debbie
TG02
Helllloooo to everyone!
For the past one week i've been in DNA diagnostic and research lab.
it was quite interesting over there. I learnt about different kinds of genetic diseases, mainly thalassemia.
In DDRL lab, alot of DNA extraction is done and majority of their samples are blood.
Hence, i did DNA extraction and that is what i'm going to post this week.
Firstly cell lysis has to be done.
1. 3 ml of whole blood has to be added to 9ml of RBC lysis solution.It is inverted to mix well and incubated for 5 mins at room temperature.
2. it should be centrifuged at 2000rcf for 2min to pellet the white blood cells.
3.The RBC lysis supernatent is then poured off and the tube is then inverted on a clean absorbent paper for 10 seconds to drain the residual liquid.
4. The tube is then vortexed vigourously to resuspend the cells in the residual liquid.
5. 3ml of Cell lysis solution is added to resuspend the cells in the tube.
6. To lyse the cells, the tube is vortexed on high speed for 10 seconds.
Next protein precipitation has to be done.
1. 1ml of protein precipitation solution is added to the cell lysate.
2. The tube is vortexed at high speed for 20 seconds to mix the protein precipitation solution uniformly.
3. It is then centrifuged at 2000rcf for 5 min. This is to precipitate the proteins
to create a tight, brown pellet.
Following that DNA precipitation has to be done.
1. The supernatant containing the DNA ( leaving behind the precipitated protein pellet) is being poured into a 15ml centrifuge tube containing 3ml 100% Isopropanol
2. the sample are mixed by inverting until the white threads of DNA are being seen.
3. The tube is centrifuged for 3min at 2000rcf, where the DNA will be visible as a small white pellet.
4. The supernatant is poured off and the tube is drained by inverting the tube on a clean absorbent paper. 70% ethanol is added and inverted several times to wash the DNA.
5. The tube is then centrifuged at 2000rcf and then the ethanol is poured off carefully.
6. The tube is then inverted to drain on a clean absorbent paper, and allowed to air dry for 15 mins.
The last step is DNA hydration.
1. 250 microlitre of DNA hydration solution is then added.
2. Sample could be rehydrated by storing at 65 degree celcius for 1 hr or room temperature overnight.
3. the sample is then transferred to a ependorff tube and stored at 4 degree celcius.
This is the whole process of DNA extraction from blood.Its quite technical. If u guys want more explanation, pls do ask me.
Thanks and have fun!
Neela
TG02
Hey to all,
http://science9.files.wordpress.com/2007/04/diabetes-glucose.gif
Both hyperglycemia (increase in blood glucose level) and hypoglycemia (decrease in blood glucose level) could be caused by renal dieases.
As O2 consumption is measured instead of peroxide formation, peroxide must be destroyed without causing release of oxygen. 
Therefore ethanol is added in the presence of catalase.
To ensure complete destruction of peroxide, iodide and molybdate are added to the enzyme reagent, causing the following reaction:
- Last week review...
ResultPicture 1
This week I am going to share on how to get a recombinant Bacmid and how to package this recombinant Bacmid into a virus.
This will require 2 steps, Transformation into a special cell call 10a and Transfection into host cells respectively.
Method is just some common protocol, whats more impt is the principle =)
Transformation
Method
1. Obtain the 10a competent E.coli cells from -80ºC freezer (1 vial = 100μl) and carry it in ice box. Thaw the cells on ice.
2. Aliquot the appropriate amount of recombinant plasmid DNA into an eppendorf tube.
3. Place the tube onto ice.
4. Aliquot 25μl of 10a competent E.coli cells into the tube ON ICE, and incubate for ½ hour.
5. Meanwhile, check that antibiotic plates (Kanamycin, Getamycin, Tetracycline, Bluo-gal, IPTG) are warmed up in the 37°C incubator (plates are inverted to prevent contamination) and ensure that water bath is heated up to 42°C
6. After the incubation for ½ hour, heat shock the bacteria cells for exactly 45 seconds in 42°C water bath without shaking.
7. Immediately transfer it onto ice for 1 to 2 mins
8. In the LAF hood, add S.O.C medium at room temperature (similar to LB medium) in 1:9 ratio to competent cells. è therefore add 225μl of S.O.C medium into each tube.
9. Shake the tube at 37°C at 300rpm in an incubator for 4 hour.
10. Obtained the transformed cells from the incubator and place it in the LAF hood.
11. Using a sterile disposable spreader, plate the whole volume (250 µl) of the each tube of cells onto the pre-warmed antibiotic plate.
12. Allow both plates to stand in the LAF hood with covers fully covered for 1 to 2 mins to allow E.coli cells to attach to agar.
13. Invert the plates (to prevent contamination) and put it into the 37ºC incubator. (incubate for 24 hours to see colonies and 48hours to see blue and white colonies)
14. After 48 hours, obtain the plate from the incubator, parafilm it and store in 4ºC fridge.
Principle
The gene of interest in the recombinant plasmid is flanked by the left and right arms of Tn7 and contains ampicillin and gentamycin resistance gene for selection, and other features to form a mini Tn7. (Shown in picture 1-above)
Host cell for this plasmid, 10a cells, contains a bacmid with a mini-attTn7 target site and a helper plasmid. When recombinant combinant plasmid was transformed into the 10a cells, transposition between the mini Tn7 on the plasmid and mini-attTn7 target site on the Bacmid occurred to produce a recombinant Bacmid. (transposition is something like “transfer” the recombinant plasmid into the Bacmid).
Recombinant bacmid was selected out on specific antibiotics plates containing (tetracycline, gentamycin, kanamycin, IPTG and bluo-gal), restreaked on new antibiotic plates, PCR and analyzed, followed by extraction and purification using the Miniprep kit.Tetracycline – because of tetracycline gene in the helper plasmid
Gentamycin- because of gentamycin gene found in recombinant plasmid
Kanamycin- because of kanamycin gene found int the Bacmid
IPTG & bluo-gal- For blue-white selection. Presence of recombinant plasmid in bacmid (white). No recombinant plasmid in bacmid (Blue).
Transfection
Method
*transfection can cause alot of cells to die, as the DNA:lipid complexes complex that are to be taken up by the cells are toxic, therefore to ensure we get as much transfected cells as possible we have to be very careful and ensure the conditions are optimal.
1. In a 6-well culture plate, seed 4 x 105 host cells/ml per well in 2ml of supplemented medium (medium suitable for culturing the host cells)
2. Allow the cells to attach at 27°C for at least 1 hour.
3. After about 15 mins later, obtain the unsupplemented medium (free of serum) from the fridge and a new 50ml tube and place it in the BSC.
4. In the BSC, aliquot about 12ml of unsupplemented medium into a new sterile 50ml tube, and return the original bottle back into the fridge.
5. We have to prepare 2 eppendorf tubes, 1 eppedorf tube for the cellfectin reagent and the other for the recombinant Bacmid DNA.
6. In the BSC, prepare as follows in 1.5ml eppendorf tubes:
Tube 1: Dilute 1µg (~0.7µl of our sample) of each purified bacmid DNA in 100µl of unsupplemented medium. (Important: ONLY pipette 1 time)
Tube 2: Mix cellfectin® reagent thoroughly before use by inverting the main tube 5-10 times. Remove 6µl of cellfectin® reagent and dilute in 100µl of unsupplemented medium.
Tube 3: Combine 100µl of the diluted Cellfectin® Reagent to tube of sample ( ~210µl). Mix gently by just tapping the bottom of the bottle, and incubate for 30minutes at RT in the BSC.
7. By now the cells should have incubated for about ½ hour. Obtain the cells on the 6 well plate from the incubator and check under the phase contrast microscope that the cells has already attached to the surface of the 6 well plate.
8. While the DNA:lipid complexes are incubating, remove the medium from the cells in the 6 well plate and wash once with 2ml of unsupplemented medium. Do this by pipetting away the old medium and replace it with 2ml of
unsupplemented medium and swirl the plate to make sure that the cells are washed. Then return the plate into the incubator.
9. After the ½ hour incubation of the Bacmid DNA: Cellfectin® Reagent complex in the BSC, take out the 6 well plate from the incubator and introduce it into the BSC.
10. Pipette out the 2ml of unsupplemented medium and add 0.8ml of unsupplemented medium to each well, followed by 200µl of the Bacmid DNA: Cellfectin® Reagent complex, swirl and rock the 6 well plate after adding.
11. Label the wells according to what was added and incubate in the 27°C incubator for 5 hours.
After about 4 hours of incubation, prepare unsupplemented medium containing antibioticsà 50units/ml penicillin and 50µg/ml streptomycin final concentration
12. After 5 hours, remove the Bacmid DNA: Cellfectin® Reagent complex from the well and add 2ml of supplemented medium to cells.
13. Incubate the transfected cells in the 27°C incubator for 72 hours or until we start to see signs of viral infections.
Principle
Purified recombinant bacmid were complex with cellfectin® reagent and transfected into host cells. (how cellfectin reagent work is already shared in week 1). This complex will cause formation of liposomal structure that causes host cells to uptake the complex.
The original bacmid before transformation was done, is actually a modified genome of the virus (got lac Z and antibiotic genes etc). The recombinant bacmid is the genome of the virus, now consisting of the recombinant plasmid in its Tn7 site.
After about 4 days, signs of viral infection start to appear, and viruses start to bud out.Signs of infect:
1. Black dots in cell nucleus
2. Cells look swollen
3. Rough surfaces of cells
4. So cells will look fused together
Preparation of the supplemented medium containing antibioticsThe purpose of adding antibiotics to this medium that the cells are going to be incubated in for 72 hours is to prevent bacteria contamination. Bacmid is a piece of very large DNA, and since it is left as a DNA in the naked form to be incubated with the cells, it can easily be contaminated by Bacteria, to prevent this from happening we added this 2 antibiotic as recommended by the manufacturers.
we usually do about 200 folds of dilution
Therefore, since in total we prepare about 40ml of the supplemented medium in this case, and antibiotic mixture (200µl) and pipette up and down to mix thoroughly.
Supplemented and unsupplemented medium
For supplemented medium, we have to add supplement such as FBS (Fetal Bovine Serum). Serum is expensive and also needs to be purified from the medium if the DNA is to be used for transfection, as the serum can interfere with transfection and results in lower efficiency. But serum allows the cells to grow better as it provide more nutrient than unsupplemented medium. Unsupplemented medium is only use for transfection.
Viruses are collected in an eppendorf tube and stored at 4 degree celcius fridge. These viruses are amplified to produce second and third batch of viruses to be used (will be shared in next post). To check for the titer of the viruses constructed, plaque assays are done. (shared in post 1)
Thanks for reading this very long post n not v nice pics i noe.. hahas..cos its all molecular stuff, i cant really show u guys the real thing =/ Hope u all understand what i have shared.Feel free to ask qns. Enjoy ur attachment =)
Jean Leong
TG02