agorganic

G'day,

I'm working on my research project which formed part of a thesis and need to spend a few days of the next week or so to finish up the last phase of the laboratory work by adding the restriction enzymes to determine whether or not a specific gene is present in my samples.

I have 70 gDNA samples (from ewes each bearing a single foetus), although I made an error in pippetting the gDNA in about half (33) the PCR plate wells and rather than adding 5ul, I ended up adding 50 microlitres of gDNA in with the PCR master mix 1 (made up of redtaq polymerase/primer/buffer/H20 mix, which 45ul is added into each well of the PCR plate). Therefore there is a total volume of 95ul in each well for half (33) of the samples and I've used up a significant amount of the Redtaq in stock, so can't go back and repeat these. The PCR wells with the correct volume have 5ul of gDNA along with 45ul of the master mix 1.

Yet to be made is Master mix 2 for my restriction enzymes (AVAII), which the documentation is attached.

A lab tech who has been assisting suggested making the master mix into a new PCR plate using the following method:

5ul of PCR master mix 1 (gDNA) into each well and resuspend
2ul of restriction enzyme
3ul of buffer
15ul of H20

My project supervisor suggested the restriction enzyme would be too much, if I can conserve it without influencing my downstream analysis, then that would be great. Except I'm not overly familiar with this and time for error is closing fast. If anyone has any other suggestions (calculations factoring in a couple of ghost samples for spares).

Depending on how long this takes me tomorrow morning, I might initially run 12 samples with the master mix 2 and put into the thermocycler, although my supervisor recommended to run the program for as long as I could. The thermocycler will sit at 12 degrees C when complete. Then I'll run twelve samples on the agarose gel, but I've got at least 34 samples out of 70 that I could test. 2 of the 70 were intended as dirty [whole blood] PCR and hadn't shown up in my gel electrophoresis, having wanted to use kangaroo (sex unkown) and a sheep from a different farm as controls.

I'll run a ladder, which is quite big, though something to compare with; along with a non-digested enzyme, gDNA, PCR (master mix 1 only) and then the digested enzymes master mix 2 samples.

If someone could provide feedback on what master mix to use instead of the one above, that is in line with the AVAII restriction enzyme procedure, then that would be great.

Jesse

Hi there,

Questions: What type of genetic database would suit my application? And how would I go about writing it up to include in my thesis?

I seem to recall there being a site where you can upload/enter your DNA absorbance data, does anyone know of where I can do this?

Background:

I am a 3rd year degree student studying a bachelor of Agriculture and compiling the final touches to my thesis which involves correlations between genetics and metabolic parameters.

I have extracted gDNA from 63 whole blood samples withdrawn from pregnant ewes bearing single foetuses, I used a Qiagen Flexigene DNA kit for the extraction process.

I am looking for the Booroola fecundity gene or Bone morphogenetic protein receptor type-1B (BMPR1B), in association with glucose and ketone measurements taken using the Abbott exceed human diabetic glucometer, the latter part already undertaken and only awaiting on my project supervisor to quide me with the PCR and amplification with the forward and reverse primers we have.

The DNA extraction was initially undertaken on 24 samples, as the centrifuge spacing was the limiting factor, although the test was ran 4 times and therefore I have numerous amounts of DNA extracted on these 24 samples. The remaining 35 samples only had the DNA extracted once on the final Test 4 (being an extension).

I've come up against numerous obstacles (too many to list), as such I've needed to cut short the research project and finalise the thesis for submission over the following week without having completed the research project, although once my exams are over the university is willing to allow me access to the laboratory to complete the research project to enable me to gain the experience in genetics I desire, as I've not been formally trained in genetics. It is my intention to submit a supplementary report ontop of the submitted (incomplete) thesis and I would like to get some feedback and suggestions on going about this, along with your thoughts on creating a genetic database for the university as a research resource. It would be nice if I could include a small section discussing how I intend to go about this.

Presently the university don't offer any courses higher than a bachelor degree, although they are investigating offering honours in the near future, so there is potential that someone (possibly myself) will take up from where my research was left.

I still have the whole blood vials which were refrigerated at 4 degrees C over four days, then frozen in a frosting freezer at -20 degrees C for 4 months and thawed overnight at 4 degrees C, then kept refrigerated at this temperature since the 29th August 2012. I could pipette 2 mls of the whole blood and freezer these into microfuge tubes at -20 degrees C and retain the vials of blood in the frigde.

The extracted gDNA from Test 4 and Test 4 extension have always been kept in the refrigerator with several sampels kept temporarily in a cooler or within the fume hood during use for an hour all up, while the extracted gDNA from Tests 1 - 3 have been kept at room temperature and exposed to artificial and relfected (non-direct) sunlight in the laboratory since 30th August 2012, in which I have only recently placed these in the refrigerator last week.

Initially, I was planning on using whatman FTA cards to preserve the blood samples, although I'm not sure if I will have the time to order them and the expense will probably end up being out of my pocket. I still have enough protease left in the flexigene DNA to run several more tests if needed using the lowest volume of blood, although more protease could be purchased separately at the end of the day, as there is still plenty of buffers left over from the kit.

Practically, I have analysed all the Tests (1 - 4 & T4 extension) for DNA purity with the spectrophotometer and this information is contained within an Excel master file.

My thesis (about 10mb), as it stands is accessible as download in the link to give you a more comprehensive idea and understanding of the background, which I'm currently working on the results. I had applied for an extension to the 23rd November, which I was initially granted although the extension was revised and is now due this coming monday with 5% deducted for each day overdue, which I am seriously considering holding off a day or two. Except I have 2 exams next week and would like to study so I can pass, considering this is my last year.

http://www.sendspace...MEUQ+WII9i8v2NQ

Hi there,

I'm an agriculture undergraduate in 3rd yr and just completed the a DNA extraction on about 60 samples using a Qiagen flexigene DNA kit and looking to get some tips for the next phase which is PCR. Basically I am looking for the Booroola gene in the samples.

I've got my two primers for the gene and the redtaq polymerase...the university has a thermocycler and the agarose gel equipment.

Below (or attached) is an excel file with the calculations my supevisor has provided me on one sheet, the other sheet is the results of the UV absorbance using a Cecil spectrophotometer which shows the purity of the DNA samples, which 1.8 is the goal though as I'm only after 140kb then I should have plenty of DNA based on the UV 260nm specs (see Table 5a in the excel file sheet titled 'Master mix calculations').

 Phase 2 - Gene search.draft 1.xlsx   92.29K   98 downloads

Hi there,

I'm wondering how to work out the dilution factor for

20 microliters (ul) in 2000 microliters (ul)

50 ul in 2 milliliters (ml)

100 ul in 2 ml

and

150 ul in 2 ml

In case I don't understand ur calculation, please put the dilution factors as well. I'm getting fairly tired and have alot of work ahead that is sapping my little mental strength that remains.

G'day there,

I'm working on my research project for an undergraduate course in agriculture at Northern Melbourne Institute of TAFE. I'll spare you the background, basically extracting sheep dna from 24 samples using a Flexigene DNA kit, blood was collected in EDTA tubes stored @ 4 degrees (d) celcius © over 4 days, then placed into a frosting freezer @ -20 d C since late april 2012.

Brief background on my blood samples storage: A portion of the blood samples (24) were traceable to individual animals National Livestock Identification System ear tag, these were inverted 10 times to throughly mix the blood prior to freezing to ensure the sample was frozen as whole blood; while a portion of the remaining samples that weren't traceable were allowed to remain separated into its plasma and haemoglobin constituteunts (hope thats the right tech terms) and frozen.  

All the samples were taken from the freezer last week and placed into a refrigerator at 4 d C to thaw slowly overnight. The next day I did a practice run on two blood samples (link below), which worked fine i.e. DNA precipitated into the FG3 buffer. One sample was blood that was mixed before freezing at -20 d C and mixed after thawing. While the other sample had separated into the plasma at top before freezing and I have left these samples unmixed, as I decided to experiment with 3 individual samples (i.e. # 2, 3, 4 as shown in linked photos) to see if I could extract DNA from the plasma (#2), the middle layer (#3 i.e. bottom mix of plasma and top layer of blood combined) and a sample from the base of the vial (#4).

See my photos (excludes image of large EDTA vial containing mixed whole blood, which just looks like normal)

Anyway. I've got to the end of the protocol included with the kit, using the one on page 14 based on 500 microlitres (ul) of whole blood. Instead of isopropanol, I used ethanol (100%) and doubled the rate to 300ul.

The practice run using the 4 test samples went fine without any problems. However when I ran the actual samples none of the 24 DNA samples precipitated into the buffer.

I've added extra FG3 buffer, the protocol lists 200ul FG3, which I increased to 300ul and found this to be insufficient. I incubated for a further hour at 65 d C in a water bath, with no success except for one sample that still had DNA particles visible to the naked eye.

Then I incubated overnight at 55 d C for 20 hours and two more samples were partially dissolved with small particles still visible to naked eye.

I placed the samples in a device with a rotating platform that heats to 60 d C and left for an hour, though no change occurred.

I tried to vortex several samples at the maximum vortex speed to break up the DNA by sheer force and some of the samples containing lighter DNA partially dissolved though not completely and the darker DNA samples were practically unbreakable. Given one sample was being vortexed for around 2 mins and the supernatant (FG3 buffer) was able to penetrate between the lid and force its way out of the microfuge tube, I figured a contamination issue may entail and had to change my gloves between samples, so I gave up on this idea. If I didn't hold the tube sideways, I could control these issue, though it meant the process would take much longer. Time is not on my side.

Next I put the samples into a heat incubator oven for an hour at 60 d C. This didn't work either, the DNA was still present and just as tough to dissolve.

Then I vortexed all the samples for 15 mins at 17,000 x g 15 d C, which allowed the DNA to stick to the bottom of the microfuge tube, though requires careful handling not to dislodge as I wanted to discard some of the supernatant from a couple of samples for further experimental testing. See below.

Following I took 8 samples and split them into two groups (see image of the hand written table and notes), note: these samples are not shown in the images linked to this discussion. In group 1, an additional volume of FG 3 buffer was added, this meant two samples had 400ul and the other two had 500ul. Out of the group 1 microfuge tube samples I labelled one 400ul sample 'V' (for vortex) and one 500ul sample 'V', the other two I will as non-vortex. This is clearly demonstrated in the image of the table (left side) I hand wrote as a visual aide.

The samples in group 2 on the otherhand, I decided to discard the FG 3 buffer supernatant and replace with fresh FG 3 buffer, see image of hand written table (right side) titled 'FG# replaced with fresh FG3 (total)', again two samples were vortexed as they were for group 1.

Interestingly enough, the group 2 fresh FG 3 buffer replacement together with vortexing did break up the dna and it was largely dissolved, though still minor particles visible to naked eye. The group 1 samples that I vortex, did seem to break up DNA better when the volume of additional FG3 was 500 ul, though it took longer to break up and more effort on the vortex than group 2 samples which were vortexed.

I placed these eight samples in the incubator heat oven at 60 d C (hottest temp.), which at present has been 1 hour and 30 mins. I am going to vortex both group 1 and 2 then go from there.

Any comments, feedback, suggestions or references please let me know.

Regards,

Jess Hughes