12 replies to this topic

[hidayahwannur]

bad gel?

[HOYAJM]

Yes a lot of those gels are not good. But the fourth gel from the left has defined bands and depending what size the plasmid is, those could be your plasmid. From a good prep, most of the DNA should be supercoiled plasmid that will migrate further than expected. Nicked/relaxed DNA will hardly migrate and I think that might be what you are seeing in the fourth gel since you have two bands. The rest of the gels are hard to make out what I am looking at.

[bob1]

You have overloaded or underloaded, understained and have heavy ethidium shadow in most of your gels.

[hidayahwannur]

HOYAJM, on 18 February 2012 - 07:49 AM, said:

Yes a lot of those gels are not good. But the fourth gel from the left has defined bands and depending what size the plasmid is, those could be your plasmid. From a good prep, most of the DNA should be supercoiled plasmid that will migrate further than expected. Nicked/relaxed DNA will hardly migrate and I think that might be what you are seeing in the fourth gel since you have two bands. The rest of the gels are hard to make out what I am looking at.

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?

[hidayahwannur]

bob1, on 18 February 2012 - 04:06 PM, said:

You have overloaded or underloaded, understained and have heavy ethidium shadow in most of your gels.

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.

[Evanescence]

The best way to do plasmid extraction is using Alkaline lysis (solution I,II,III) however if you just need to screen the transformant i think TENS solution is faster... the advantage of using kits is the column, doing traditional one need you to use phenol chloroform which is labor extensive...but for me if you want to recycle the column it's okay as long as the plasmid you want to extract is same

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,
Evanescence

[bob1]

hidayahwannur, on 18 February 2012 - 05:45 PM, said:

bob1, on 18 February 2012 - 04:06 PM, said:

You have overloaded or underloaded, understained and have heavy ethidium shadow in most of your gels.

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.

First off you need to choose a % gel that is suited to your application - a 1% gel will separate bands from 500 bp up to 10 kbp, however a 1.5% gel will separate 250 bp - 5 kbp better and a 2% will separate 100 bp to 2.5 kbp nicely.  Next is to make the gel, as you would normally.  Loading - you need to load in the range of 50 ng per millimeter of well width for ladders (i.e. for a 5 mm wide well, add 250 ng), but this varies as the DNA size varies - larger products will intercalate more EtBr and so be brighter on a gel than shorter products.

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

[hidayahwannur]

bob1, on 19 February 2012 - 12:15 PM, said:

hidayahwannur, on 18 February 2012 - 05:45 PM, said:

bob1, on 18 February 2012 - 04:06 PM, said:

You have overloaded or underloaded, understained and have heavy ethidium shadow in most of your gels.

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.

First off you need to choose a % gel that is suited to your application - a 1% gel will separate bands from 500 bp up to 10 kbp, however a 1.5% gel will separate 250 bp - 5 kbp better and a 2% will separate 100 bp to 2.5 kbp nicely. Next is to make the gel, as you would normally. Loading - you need to load in the range of 50 ng per millimeter of well width for ladders (i.e. for a 5 mm wide well, add 250 ng), but this varies as the DNA size varies - larger products will intercalate more EtBr and so be brighter on a gel than shorter products. 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

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

[phage434]

A microwave works better than an oven.  The solution should be completely clear and well mixed after heating.  I never noticed that it was necessary to cool the solution prior to adding EtBr.  The gel should be allowed to cool thoroughly.  You should add similarly small amounts of EtBr to the positive buffer compartment of the tray (step 8a) after filling to just above the height of the gel.  I would recommend a loading buffer containing orange-G instead of the blue dyes.  My favorite is 15% ficoll + orange G, use at 5x.

[hidayahwannur]

phage434, on 20 February 2012 - 05:40 AM, said:

A microwave works better than an oven. The solution should be completely clear and well mixed after heating. I never noticed that it was necessary to cool the solution prior to adding EtBr. The gel should be allowed to cool thoroughly. You should add similarly small amounts of EtBr to the positive buffer compartment of the tray (step 8a) after filling to just above the height of the gel. I would recommend a loading buffer containing orange-G instead of the blue dyes. My favorite is 15% ficoll + orange G, use at 5x.

do u mean i need to add Etbr after i filled the tank containing the solidify gel? not after the heating process?

[phage434]

Both.

[hidayahwannur]

SO I NEED TO ADD 2 TIMES?

[phage434]

Add some EtBr to the agarose while it is hot and before pouring the gel.  Add some EtBr to the buffer at the positive end of your tray before running your gel.