[Private] The methods I used

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bbs_r
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[Private] The methods I used

Post by bbs_r » Tue Apr 04, 2006 7:12 am

Experiment 2: PCR product extraction
After electrophoresis, a long wavelength UV machine was used to locate the target molecular bands in the agarose gel. The region containing the target bands (<600 bps) was isolated by a clean sharp blade and was transferred to a 1.5 mL microfuge tube. Weight of the gel was obtained via comparing to an empty microfuge tube (first trial: 170mg). 3 volumes of buffer QX1 was added to 1 volume of gel (first trial: 510 μL). The mixture was vortexed for 30 secs and then 10 μL of QIAEX II particles were added.
The sample mixture was incubated at 50˚C for exactly 10 mins and vortexed every 2 mins. After that, the sample was centrifuged for 30 secs and the supernatant was removed carefully. The pellet was washed with 500 μL of buffer QX1, followed by vortexing, 30 secs centrifugation and the removal of supernatant. The pellet washing procedure was repeated twice with buffer PE instead of buffer QX1. Finally, the pellet, which contains the cDNA extract, was air-dried for 10-15 mins.
The pellet was resuspended in 23 μL of distilled water in the microfuge tube by vortexing. After that the mixture was centrifuged for 30 secs and the supernatant, which contains the purified cDNA, was carefully transferred to a new microfuge tube.


Experiment 3a: cDNA cloning
Transformation mixture preparation

One clean 0.5 mL eppendorf tube was used, in which 3 μL of sterile water was added and then followed by 3 μL of PCR product, 1 μL of 10X ligation buffer, 2 μL of pCRII vector (25 ng/μL) and finally 1 μL of T4 DNA ligase (4 units). After that, the mixture was incubated at 14˚C for overnight.

Competent Cells Transformation
The incubated mixture was centrifuged briefly and was placed on ice together with one tube of 50 μL TOP10F’ (One Shot) Competent cells. Then, 2 μL of ligation product was transferred to the TOP10F’ cells and was mixed by stirring gently with pipette tip. The TOP10F’ cell mixture was incubated on ice for 30 minutes. After that, a heat shock was given at 42˚C for exactly 30 secs without shaking and then the TOP10F’ mixture was immediately transferred back on ice. 250 μL of SOC medium was added to the TOP10F’ cell mixture and it was shook horizontally at 37˚C for 1 hour at 225 rpm in a shaking incubator.
10 μL of the shook culture was transferred to a clean tube containing 90 μL of SOC medium (i.e. 1 in 10 dilutions). Both original culture and diluted culture were spread evenly on separate LB agar plates containing 50 μg/mL of kanamycin, 10 μL of X-Gal and 1 μL of IPTG. These plates were then incubated overnight at 37˚C.

Transformant Cloning
10 white colonies were selected from the LB agar plate. Each colony was inoculated to separate clean culture tubes containing 2 mL of LB broth and 2 μL of kanamycin. The cultures were stored in a vigorous shaking incubator for 16 hours.


Experiment 3b: Transformant Analysis
cDNA Extraction from the Transformant

The incubated cultures were transferred to separate clean 1.5 mL microfuge tubes and centrifuged at 13000 rpm in a table-top microcentrifuge for 3 mins at room temperature. For each microfuge tube, all supernatants were removed. 250 μL of buffer P1 was added to resuspend the pellet. After that, 250 μL of buffer P2 was added to the mixture and mixed thoroughly by inverting the tube 6 times. The same step was repeated once with 350 μL of buffer N3 instead of buffer P2. Then, the mixture was centrifuged for 10 mins at 13000 rpm in a table-top microcentrifuge.
The supernatants from different cultures were transferred to separate spin columns by pipetting and the vacuum source was switched on. Once the solution was drawn through the column, the vacuum source was switched off. 0.5 mL of buffer PB was added to wash the spin columns and the vacuum source was switched on again until the solution was drawn through the column. The same step was repeated by adding 0.75 mL of buffer PE instead of buffer PB. The spin columns were centrifuged for 1 min and then transferred to separate clean microfuge tubes. Finally, 50 μL of buffer EB (distilled water) was added at the center of spin column and let stand for 1 min to elute DNA. Then, the spin columns were centrifuged for 1 min and the solution collected at the microfuge tubes contained the eluted DNA.

Restriction Enzyme Digest
1 μL of cDNA sample was mixed with 1 μL of NE buffer 2 (10x), 1 μL of ECoRI and 7 μL of distilled water. The mixture was put in a 37˚C water bath for 1 hour and it was analysed by agarose gel electrophoresis.

Experiment 4: cDNA sequencing
Cycle Sequencing

1 μL of cDNA sample was mixed with 14.8 μL of distilled water, 1.2 μL of buffer (5X), 1 μL of T7 primer (40 μg/μL) and 2 μL of Terminator Ready Reaction Mix. The mixture was placed in a GeneAmp PCR System 9600 with settings shown in table 4.
Control was done by using pGEM-3Zf (+) template and -21 M13 forward primer.

Purifying Extension Products
20 μL of extracted transformant cDNA was mixed with 5 μL of Sodium Acetate (pH 4.6) in a 1.5 mL microfuge tube. 110 μL of 100% ethanol (2 volumes) was added to the mixture. The mixture was vortexed and centrifuged at 14000 rpm for 30 minutes at 4 ºC. Supernatant was removed and the pellet was washed by adding 250 μL of 70% Ethanol. The mixture was cold centrifuged at 14000 rpm for 30 minutes again and all supernatant was removed. Finally, the pellet was leaved on ice and air dried.

cDNA sequence analysis by ABI PRISM 310
The sample was resuspended in 25 μL of Template Suppression Reagent (P/N 401674) and vortexed. Then, the sample was heated at 95˚C for 2 mins to denature and vortexed. After that, the sample was transferred to the sequencer and the result was obtained in the next morning.

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Post by bbs_r » Tue Apr 04, 2006 7:14 am

This is what I wrote on my dissertation. Do I give too much details? However, my objective in the project is just to:

1) confirm the presence of VAP protein by PCR
2) sequence VAP protein

Nothing more than that...

Since I really need a 2:1 for future studies, I beg for your assistance.

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Post by bbs_r » Tue Apr 04, 2006 7:24 am

By the way, for the pGEM-3Zf (+) template and M13 control, do I need to demonstrate my control sequence in the dissertation?

Thank you very much for your help once again!

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Post by canalon » Tue Apr 04, 2006 11:51 am

You do not give anyhing about step 1 are you doing a RT_PCR (using RNA as template) or not? Because if as it seems you do not, you should not talk about cDNA...

In the last step, what you are doing is not perfectly clear. The mixture you are purifiying is the cycle sequencing mixture not extracted transformants cDNA. And from what I read 110 µl are not 2 volumes when compared to 20+5 µl. And the pellet was left on ice, ot leaved on ice.

Ah and for the transformant cloning, cells are not stored! but grown, incubated...

Now for the level of detail, I don't really know since it also depends on your teacher. In a scientific paper this would be way too detailed, but I made somr dissertation where this would be OK.

I would say that you do not need to show your controls results, it is usally enough to say what you used as a control, ans everybody will assume they are OK. But if you feel inconfortable with this, just put the sequence in an appendix or something like that.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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Post by bbs_r » Thu Apr 06, 2006 9:42 am

Canalon wrote:You do not give anyhing about step 1 are you doing a RT_PCR (using RNA as template) or not? Because if as it seems you do not, you should not talk about cDNA...


Since the step 1 is more complicated than usual, I haven't write it up yet. This is why I didn't include my Step 1 in here. By the way, I used a Touch-down PCR in this case.

In fact, I think I need a hand about this matter, too. What I did in step 1 is:
------------------------------
I run a PCR with a particular given recipe, it did not work. Then, I changed recipe a wee bit, but it was still not working. Then, I changed the recipe again and I also changed the PCR temp and cycle a bit. It was not working as well. Then, I changed the recipe again, not work. I changed the PCR temp and cycle a bit, not work. Finally, my supervisor shown me how to pipette the material. It works but only for the control. I changed the recipe again with DNA precipitation before mixing things up, it works then.
------------------------------
To write the above things up in the material and methods, I found it is VERY DIFFICULT. Especially I don't know whether I should write the whole story there or not. Or I only need to write the steps up with the final recipe? Since my supervisor mentioned the photos I took for the electrophoresis are used to write my troubleshooting up. I REALLY REALLY don't know whether this means I need to write the story from the very beginning or not, if so, those steps at the beginning would involved too many "Then" and "After that", which makes the reading a bit "bad".

May you give me a hand on this matter please? Thank you again!

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