GDSYZX-United/HUMAN PRACTICE/human practice

Extract DNA in arabidopsis thaliana

Place:Biology Experiment Teaching Center Of Sun Yat-sen University

Date:July 25th, 2016

Experimental apparatus:Grinding rods, 2 ml centrifuge tube, double panel, transfer liquid gun, adsorption column &collection tube and centrifuge.

Experimental drugs:Buffer PCB, β- mercaptoethanol, RNaseA, chloroform, Buffer BD, anhydrous ethanol, PW Solution, Wash Solution, TE Buffer.

Procedure:

Experimental drugs	operation	Time
50-100mg Young leaves of Arabidopsis thaliana.	Transfer to 2 ml centrifuge tube after grinding with grinding bar.
600μl 65℃ Buffer PCB & 12μl β- mercaptoethanol	Placed in 65 ℃ water after blending.	25min
20μl RNaseA	waiting	5min
About 630μl chloroform	Transfer supernatant liquid to a clean 2 ml centrifuge tube after blending and following 12000 RPM centrifuge.	centrifuge：5min
About 400μl Buffer BD	Blending for 3-5 times.
About 400μl anhydrous ethanol	Transfer to the adsorption column after blending,and then waiting. Dump the waste liquid after centrifuging with 10000 RPM.	waiting：2min centrifuging：1min
500μl Wash Solution	Centrifuging with 10000 RPM after adding the reagent, and then dump the waste liquid.	1min
	centrifuge with 12000 RPM.	2min
50μl TE Buffer	Place the adsorption column in the new 2 ml centrifuge tube, then waiting after adding reagent.	wait:3min
	centrifuge with 12000 RPM.	2min

Result:

The target DNA can be extracted successfully in most of the groups, but they have very low concentration and many impurities.

Introspection:

1. Understanding of the experimental requirements was not very well and had some wrong operations in the experiment.
2. There is still many liquid residue on the grinding rod after grinding Arabidopsis thaliana leaves.
3. Not all reagents were fully blended.
4. The usage of transfer liquid gun was not normative.

arabidopsis thaliana

grinding with grinding bar

After blending with Buffer PCB

centrifuge

result