seeking cloning info - (Jul/21/2006 )

hi all

would anyone know if:

1. xbaI shows star activity or not
2. overnight digestion with it is good, or should i restrict it to 3-5 h
3. how long to expose for dephosphorylation with calf intestial phosphatase

thanks

viv

hi
1 - no star activity
2 - if you wonder oof that, use a pcr program with 37° for 6h
3 - 1h at least.

Agree with Fred.

I digest for 2-3 hrs only with xbaI.

Neb's webpage is one of the more useful tools ever for questions regarding restriction enzymes; for specifics you may also look up the individual enzyme itself

try this link

thanks fred, scolix and aimikins

will post the result info soon as i finish this

- viv

hi you three

ok, so i digested pUC 19 with xbaI for 4 hours at 37 C, but when i did the gel to gel purify digested vector for ligation, i found there were 4 bands: two very close almost making a duplex, and one a little higher and the fourth still higher up.

what do you think??????

it would be good if you could post a picture?

also, have you tested your enzymes against any other DNA? have they been stored properly? what about the buffers and BSA?

well picture should help...
try to digest by an other enzyme to check of the good vector.
At least do you have a band at expected size?

i did not keep a picture of the gel, so cannot upload. but i guess the problem was that there was too much vector against the restriction enzyme. the puc dna was good as it was bought first hand from fermentas, but i used 1 µg with 1 µl (10 u) of xbaI. that was when there were those four bands.

i then used the dna to transform bacteria (essentially to make my own stock), and did a miniprep using qiagen kit. i then used 1 µl of this and digested it with 1 µl xbaI. aliquots were withdrawn every hour and stored. the digestion was allowed to proceed overnight, and then the next morning, a gel was run.

all the aliquots showed a single clean band, which means that even a 1 hour digestion (the minimum i tested) is good.

is there some formula that calculates the number of enzyme units to be used with a fixed amount of dna?

thanks to all

- viv

according to unit definition; 1 unit of Restriction Endonuclease is the amount required to completely digest 1 ug of DNA in one hour in appropriate buffer provided.

i am generally using 1 units of enzyme per 1 micrograms of DNA substrate regardless of incubation time.