agarose gel electrophoresis of plasmid extraction
Posted 18 February 2012 - 07:49 AM
Posted 18 February 2012 - 04:06 PM
Posted 18 February 2012 - 05:40 PM
yes..and really frustrated with the result. i want to redo the plasmid extraction and hope i will get the good one. by the way, do u have method for traditional plasmid extraction?i dont want to use kit,. i want to use traditional method , alkaline lysis. may i have the method and the recipe for the media/ingredients preparation?
Posted 18 February 2012 - 05:45 PM
what should i do actually? usually, what is the accurate steps in doing the agarose? can u tell me so that i can try again. i am in the learning process.
Posted 19 February 2012 - 09:28 AM
regularly change your running buffer to get nice gel, do not run gel when it just harden wait till it ready to use, sharp band will appear for you
all the best,
Posted 19 February 2012 - 12:15 PM
You loaded EtBr into your gel. EtBr runs in the opposite direction to the DNA on the gel, so you end up with "ethidium shadow" where the EtBr has run up the gel and is no longer staining the lower portion. To avoid this, either post-stain your gel in running buffer (e.g. TBE,TAE or SB) with 0.5 ug/ml or add EtBr to your running buffer at the same concentration. The thinner your gel the better, it will give you less background.
For plasmid extractions - a classic protocol for minipreps can be found here
Posted 20 February 2012 - 03:03 AM
thank you for your information...can u please check my method..if anything i need to change, please inform me...i already extract the plasmid, tomorrow i will run the gel
1. weight 0.4g agarose gel
2. transfer into conical flask
3. add 40ml 1X TAE buffer
4.cook for 2 minutes in oven
5. let it warm , add etbr (end of tips) (better how much need to add?)
6.pour into the casting tray
7. let it solidify
8. pour 1X TAE buffer into the tank
9. load sample (5ul + 2 ul loading dye), DNA ladder 2ul (1kb & 100 bp)
10. run gel 90V, 90 minutes
Posted 20 February 2012 - 05:40 AM
Posted 20 February 2012 - 06:40 AM
do u mean i need to add Etbr after i filled the tank containing the solidify gel? not after the heating process?
Posted 20 February 2012 - 04:50 PM