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how to get sharp band for restriction enzyme


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#1 zack

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Posted 11 November 2009 - 09:02 AM

hi admin and chatter,

i would like to ask about, how to get sharp band becoz i get my restriction enzyme was not a sharp band, for your information i already use thin, thinnest well and also increase agarose gel into 2% until 3%. but still not get a sharp band.

i using 6x loading dye. my REs is mnII, hpy, cky,dde1 and total 8 re.
it is becoz of my loading dye?

thank you.

#2 skeuos

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Posted 11 November 2009 - 02:29 PM

hi admin and chatter,

i would like to ask about, how to get sharp band becoz i get my restriction enzyme was not a sharp band, for your information i already use thin, thinnest well and also increase agarose gel into 2% until 3%. but still not get a sharp band.

i using 6x loading dye. my REs is mnII, hpy, cky,dde1 and total 8 re.
it is becoz of my loading dye?

thank you.


How much DNA are you loading? Loading excess will cause streaks. Perhaps try loading only half your reaction. What are the reaction conditions (buffer, +/- BSA, temp, length of digest, etc).

#3 zack

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Posted 11 November 2009 - 07:20 PM

hi admin and chatter,

i would like to ask about, how to get sharp band becoz i get my restriction enzyme was not a sharp band, for your information i already use thin, thinnest well and also increase agarose gel into 2% until 3%. but still not get a sharp band.

i using 6x loading dye. my REs is mnII, hpy, cky,dde1 and total 8 re.
it is becoz of my loading dye?

thank you.


How much DNA are you loading? Loading excess will cause streaks. Perhaps try loading only half your reaction. What are the reaction conditions (buffer, +/- BSA, temp, length of digest, etc).


thank for reply, i;m using 6ul in 30ul total volume re reaction,

my re condition is

5ul = PCR product
3.0 = bsa
0.2 = RE
21.8 = sddh20

for 20 ul is use

5ul = PCR product
2.0 = bsa
0.2 = RE
22.8 = sddh20

length below 340




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