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troubles in DNA digestion

cloning

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7 replies to this topic

#1 LadyA2A

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Posted 03 February 2012 - 03:03 AM

Hi all,
I've been trying to digest pGEX-4T-1(4kb) and DNA sequence of Mycoplasma (514pb) using SalI and EcoRI for 4 weeks.. But i can find nothing after runnig an agarose gel (1%).
I know that 1U of the enzyme digest completly 1µg of DNA in one hour at 37°C. but I'm not able to exploit this knowledge :(
I'm using the following protocol;

H2O 5µl
OPA buffer 2x 1µl
DNA / pGEX 4µl
SalI 15U/µl 0.01µl
EcoRI15U/µl 0.01µl

I tryed diffrent times of incubation at 37°C: 60; 45 and 30 minutes.
I changed diifrent volumes of DNA.
but i still find nothing on agarose gel Posted Image

questions are:

1- How can i know if my enzymes are ok (as they ar not new)?
2- as i can't know the concentration of my plasmide neither my DNA, how can i calculate the number of unit of enzyme i'm supposed to use?

3- How can i estimate the concentration of DNA after running an agarose gel?
Has anybody had the same problems or do you have an idea what i'm doing wrong ?

( in the pic of agarose gel, i put 3 µl of pGEX as positif control : non digested plasmid)
If any one can help, I'll be really grateful Posted Image

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#2 scolix

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Posted 03 February 2012 - 01:14 PM

Its difficult to pipette 0.01ul of enzyme.
You need to have a positive control for the enzymes to know if they digest it properly.

there are ways to calculate DNA from agarose , by running different volumes of known conc. of ladder and comparing the intensity of your sample to the ladder.

I would suggest digesting much more DNA and digest it in 20-50ul

#3 phage434

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Posted 03 February 2012 - 04:52 PM

No one can pipet .01 ul of enzyme. I'd suggest that you failed to add any. As scolix suggests, do a digestion in 50 ul. I don't know what OPA buffer is, but if it is really a 2x buffer, then you added it at 10x dilution. More likely it is a 10x buffer and you wrote it incorrectly. I would add 1 ul of each enzyme.

#4 LadyA2A

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Posted 04 February 2012 - 03:13 AM

.

Edited by LadyA2A, 04 February 2012 - 05:28 AM.


#5 LadyA2A

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Posted 04 February 2012 - 05:27 AM

Its difficult to pipette 0.01ul of enzyme.
You need to have a positive control for the enzymes to know if they digest it properly.

there are ways to calculate DNA from agarose , by running different volumes of known conc. of ladder and comparing the intensity of your sample to the ladder.

I would suggest digesting much more DNA and digest it in 20-50ul

Hi
thnx everyone for your advices
yup, my bad, i was trying to put 0.15U of each enzyme.. and as adding 0.01µl is impossibe i made a dilution of the enzyme at 1/10 from wich i took 0.1µl.. i forgot to mention that.Posted Image
OPA buffer is One-Phor-All buffer from Amersham.
But I still don't understand what 's a positif control of an enzyme??!!!!!Posted Image

Edited by LadyA2A, 04 February 2012 - 05:29 AM.


#6 phage434

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Posted 04 February 2012 - 08:40 AM

You are confused. You should be using much more enzyme. Do the reaction this way:

39 ul water
4 ul of your DNA
5 ul of OPA buffer (it really is 10x, unless you have diluted it)
1 ul of SalI
1 ul of EcoRI


Mix, digest for 1 hour at 37 degrees. Yes, there is an excess of enzyme. Yes, this is important and intentional. Yes, you could do this in a lower volume, but no, that would not be a good idea.

#7 LadyA2A

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Posted 07 March 2012 - 08:12 AM

You are confused. You should be using much more enzyme. Do the reaction this way:

39 ul water
4 ul of your DNA
5 ul of OPA buffer (it really is 10x, unless you have diluted it)
1 ul of SalI
1 ul of EcoRI


Mix, digest for 1 hour at 37 degrees. Yes, there is an excess of enzyme. Yes, this is important and intentional. Yes, you could do this in a lower volume, but no, that would not be a good idea.

hey guyes,

Thank you for your responses, it was really usefull.. i used the protocol you gave to me... and it works Posted Image but the dna in the digetion product is too much diluated ... the total volume of a ligation mixture is about 20 µl Posted Image.. the problem is that i have to transform BL 21 Posted Image

#8 phage434

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Posted 07 March 2012 - 05:42 PM

You should transform into a cloning strain such as Top10 or DH5a rather than BL21. Then, prepare plasmid from the cloning strain and then transform into BL21.
You should only need about 20 ng of digested DNA in your ligation mix.





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