I have a problem in getting colony with correct insert in the vector. My vector size is 5.1kb and insert size is about 4kb. After checking my digestion product on gel, I found that the vector was not fully digested. After that I tried to prolong the digestion time from 1.5 h to 3 h, and I still can see 3 bands on gel. Can anyone suggest how can I improve my digestion to ensure a complete digestion (only 1 band of digestion product on gel)?
Thanks in advance.
Can you show us a picture with the band sizes please. I would try cutting overnight but is this a single digestion or a double digest?. Please post cutting conditions with type of buffer, amount of enzyme and template dna etc
Dear Paul Rutland,
On the gel pic; Lane 1: 1kb marker. Lane 2: undigested plasmid. Lane 3: Double digestion with Kas1 and Kpn1 RE (My target vector 5kb). Lane 4: Single digestion with Kas1. Lane 5: Single digestion with Kpn1. Lane 6: 1kb marker
At Lane 4 and 5, I can see additional bands almost similar to my undigested plasmid. That's why I suspect my double digestion was also not fully cutted although there was no visible additional bands at Lane 2.
Cutting conditions:
a) Double digestion (Kas1-Kpn1)
x ul of pDNA (1ug)
5ul Fermentas Kpn1 restriction buffer
1ul Kpn1
1ul Kas1
1ul fast Alkaline Phosphatase
y ul MilliQ to a final volume of 50ul
b) Single digestion (Kas1)
x ul of pDNA (1ug)
5ul Tanggo buffer
1ul Kas1
1ul fast Alkaline Phosphatase
y ul MilliQ to a final volume of 50ul
c) Single digestion (Kpn1)
x ul of pDNA (1ug)
5ul Fermentas Kpn1 restriction buffer
1ul Kpn1
1ul fast Alkaline Phosphatase
y ul MilliQ to a final volume of 50ul
Digest for 3h at 37C waterbath.
Hi there,
KpnI doesn't look very active on native plasmid (due to supercoiled structure?). What I would suggest is a sequential digest : KasI first to linearize the plasmid and then KpnI digest (just add the enzyme to the first reaction mix if compatible and if KasI and KpnI sites are not too close). Then gel purified.
Try to load less material onto gel and run it longer in order to get nice profiles with good resolution at the plasmid size range.
Hello,
Try different combinations of plasmid : enzyme : time and then load 2, 5, 10 and 15 microlitres product in different wells and check.
Hi everyone, thanks for your suggestion. I did serial digestion with diluted pDNA and it seems to work well so far. I got one nice band! I will continue the next step column purification and ligation. Wish me luck !!!!