تويت
Hello people ..... Because I really realize that we should improve our skills particularly our molecular techniques I have inserted this topic that I copied from my master dissertation
the first method is The extraction with Guanidinium. it is a simple method can be done manually, while the second one, which is The extraction with QIAamp MinElute Virus Spin Kit
is a commercial kit
now, you have 2 methods to extract either DNA or RNA from your clinical samples
I hope this is useful for you all
please enjoy it and feel free to ask
see you sooooon
the first method is The extraction with Guanidinium. it is a simple method can be done manually, while the second one, which is The extraction with QIAamp MinElute Virus Spin Kit
is a commercial kit
now, you have 2 methods to extract either DNA or RNA from your clinical samples
I hope this is useful for you all
please enjoy it and feel free to ask
see you sooooon
The extraction with Guanidinium method
Nine samples and three controls were extracted by the Guanidinium method. The method used was adapted from the method of Casas et al in 1995. Briefly, 50µl of each sample was mixed with 200µl of extraction buffer containing 5M GuSCN (Sigma, UK), 1M Na Citrate (Sigma, UK), 25% Sarcosyl (Sigma, UK), 0.1M Dithiothreitol DTT (Sigma, UK), Glycogen (Roche, UK) and SDW (Gibco, UK) (see the appendix). Following incubation at room temperature for 10 minutes, sodium acetate was added for each tube with good mixing to give 0.3M. Then 275µl of ice-cold is-propanol was added to each tube. After the incubation at room temperature for five minutes, the samples were pelleted by centrifugation at 12000xg for 10 minutes. The pellets were washed in 70% ethanol and were left to air dry for five minutes then suspended in 50µl of sterile distilled water (SDW).
The extraction with QIAamp MinElute Virus Spin Kit (QIAGEN)
Nine samples and three controls were extracted with QIAamp MinElute Virus Spin kit. The procedure that was applied was exactly the same procedure as in the hand book of the kit. A volume of 25µl of the Qiagen protease was pipetted into 1.5ml eppendorfs tubes. Then 200µl of each sample was added. In the control tubes 200µl of SDW was added instead of the samples. After that, 200µl of AL buffer that contains guanidine hydrochloride with carrier RNA (see the appendix for details of the preparation of the carrier RNA) was added to the mixture in the microcentrifuge tube and that was followed by 15 seconds vortexing. The mixtures were then incubated in a dry heating block at 56°C for 15 minutes (Qiagen, 2005).
The samples were briefly centrifuged to remove drops from the inside of the lid. Next, 250µl of 100% ethanol was added to each sample. The samples were then mixed thoroughly by vortexing for 15 seconds. The tubes were then centrifuged again to make sure that there were no drops inside the lids(Qiagen, 2005).
QIAamp MinElute columns were labelled with coloured labels. The samples were transferred from the microcentrifuge tubes, that had had been processed, to QIAamp MinElute columns that had been labelled. The lysate, i.e. the lysis buffer with the samples, was transferred using Gilson pipits. The microcentrifuge tubes were discarded and the QIAamp columns, which had the lysate, were put for centrifugation at 6000 xg for one minute. The lysate passed through the filter and the nucleic acids bound to the filter. The filtrate was received into the collection tube.
All collection tubes were discarded and replaced with new collection tubes. Later, 500µl of AW2 buffer was added to each QIAamp column and centrifuged at 6000xg for 1 minute. The collection tubes were removed and discarded with the filtrates. QIAamp columns were transferred to new collection tubes. 500µl of absolute ethanol was added to each QIAamp column and centrifuged at 6000xg for one minute and the collection tubes were discarded containing the filtrate (the nucleic acids were still bound to the spine). QIAamp columns were put into new collection tubes and centrifuged at 20,000xg for three minutes to dry the membrane of the columns. QIAamp columns were placed into new 2ml collection tubes and incubated at 56°C for three minutes to dry the membrane completely.
Finally, QIAamp columns were placed into clean 1.5ml microcentrifuge tubes and The samples then were eluted by addition of 150µl buffer AVE to the centre of the membrane. After one minute incubation at room temperature, the samples were then centrifuged at high speed for one minute. As result of the centrifugation the nucleic acids will be received into the tubes.
The samples were briefly centrifuged to remove drops from the inside of the lid. Next, 250µl of 100% ethanol was added to each sample. The samples were then mixed thoroughly by vortexing for 15 seconds. The tubes were then centrifuged again to make sure that there were no drops inside the lids(Qiagen, 2005).
QIAamp MinElute columns were labelled with coloured labels. The samples were transferred from the microcentrifuge tubes, that had had been processed, to QIAamp MinElute columns that had been labelled. The lysate, i.e. the lysis buffer with the samples, was transferred using Gilson pipits. The microcentrifuge tubes were discarded and the QIAamp columns, which had the lysate, were put for centrifugation at 6000 xg for one minute. The lysate passed through the filter and the nucleic acids bound to the filter. The filtrate was received into the collection tube.
All collection tubes were discarded and replaced with new collection tubes. Later, 500µl of AW2 buffer was added to each QIAamp column and centrifuged at 6000xg for 1 minute. The collection tubes were removed and discarded with the filtrates. QIAamp columns were transferred to new collection tubes. 500µl of absolute ethanol was added to each QIAamp column and centrifuged at 6000xg for one minute and the collection tubes were discarded containing the filtrate (the nucleic acids were still bound to the spine). QIAamp columns were put into new collection tubes and centrifuged at 20,000xg for three minutes to dry the membrane of the columns. QIAamp columns were placed into new 2ml collection tubes and incubated at 56°C for three minutes to dry the membrane completely.
Finally, QIAamp columns were placed into clean 1.5ml microcentrifuge tubes and The samples then were eluted by addition of 150µl buffer AVE to the centre of the membrane. After one minute incubation at room temperature, the samples were then centrifuged at high speed for one minute. As result of the centrifugation the nucleic acids will be received into the tubes.
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