I'm doing my research on moss and right now i'm doing dna extraction from moss sample. I use the Qiagen Dneasy plant mini kit but i didn't get any result. I don't know what i do wrong. I thought there might be problem with the grinding process, because in the protocol, the instruction is :
- Place the sample material (≤100 mg wet weight or ≤20 mg lyophilized tissue) into a 2 ml safe-lock microcentrifuge tube, together with a 3 mm tungsten carbide bead.
- Freeze the tubes in liquid nitrogen for 30 s. Place the tubes into the TissueLyser Adapter Set 2 x 24, and fix into the clamps of the TissueLyser.
-Immediately grind the samples for 1 min at 30 Hz.
- Disassemble the adaptor set, remove the tubes, and refreeze in liquid nitrogen for 30 s.
When using lyophilized tissue, the tubes do not need to be frozen in liquid nitrogen.
- Repeat step 4, reversing the position of the tubes within the adaptor set. Proceed
immediately to step 7.
But, at first, i only have tissuelyser machine, so i just grind the sample with the lysis buffer using the tissuelyser machine. The liquid nitrogen just arrived today. But, i did not know to carry out the supposed procedure above. How am i supposed to freeze the tube in liquid nitrogen? Hope someone can help me with this because this is my first time doing dna extraction
Dear Nur
I am not sure that fully understand Your question, but I assume its core is - how to extract DNA from moss using Qiagen kit.
In this case, before You start to follow decently simple protocol provided with kit, there are two previous steps:
A) Sample collection
B) Sample homogenization
If one or both of these steps are not preformed correctly You can have problems with DNA extraction up to not having DNA.
For sample collection, I recommend You to do the next steps:
0) Be careful working with LN2, but do not be scared. Use gloves, glasses, and do not touch frozen metal things.
1) Have liquid nitrogen in flask
2) Pre-weight DNAse free 2ml tubes and label them
3) Clean cutting tools (if you use them for moss collection)
4) Quickly cut a pice of moss, quickly place it inside the tube, close lid and immediately drop it to flask with LN2.
The whole process, after some practicing will take 15-30 seconds.
Your samples are now collected.
Homogenization is another critical step. If You do not have tissuelyser equipment, You need to think about alternative ways of mechanical grinding. Mortar and pestle? Drill? Consult with lab manager about this. The best though will be to use bead-beater tissuelyser. If You can get access to it:
- take one sample from LN2 (use long forceps)
- quickly weight it (after subtraction of the pre-weighted tube value You will know the sample weight)
- quickly place 2 beads inside
- drop it in LN2 again
- prepare all sample in this way
- pre-cool in LN2 adapter for tubes that will be used in tissuelyser
- insert the samples to this adapter, put it in device and homogenize for 30 seconds at 20-30Hz frequency
- when it is done, put the samples from adapter to LN2 flask again
- take samples one-by-one and add buffer from the kit, then follow its protocol
I believe after 2-4 samples for practicing You will get good DNA. If not, You may think to switch to manual methods of extraction, foe example, Trizol or CTAB. But I would give kit a try - it much faster and simpler usually.
@Albert Batushansky , thank you for the suggestion. I will try the method. Thank you again for the reply
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