Double Digestion and Single Digestion same result? - Confusion about Plasmid and Insert alignment (Nov/19/2005 )
Strange results forced me to seek your guidance in forum.I am working with [i]Clostridium kluevery[/i] gemome. PCR amplified product was ligated into pCR2.1 plasmid by TA cloning method. The Insert of my GENE of interest was pooled togather and ligated into pET28a at BamHI and SalI site for expression of protein.I got the construct and just before i start expression , i wanted to check my insert and its stability. When I checked the construct with BamHI and SalI restriction enzyme(double digestion), i got 2 bands and the insert at expected range.But single Digestion with either BamHI or Sal I also gives me very fade similar band at same level.Instead of single linearised band of Plasmid with insert , i get 2 bands in single digestion too. That is making me confused about my construct and its stability.Since I am using restriction enzymes for the first time and took care of all possible contamination problem.
I checked the availability of my insert using specific PCR primers and i get amplified product at expected range again .Sequence information is not yet available with me.Can you let me know why I am getting such results? Thanks in advance.
Mie University , Japan
would you please post a picture of your gel?
and would you please give a little more detail regarding your cloning strategy and how you set it up?
I suspect your insert is not what you think it is, and we need to figure out why...
How did you recover your insert from the TA cloning vector? And were there restriction sites engineered into your PCR product?
Thanks for your reply. Yes, PCR was done using KOD Dash Polymerase to facilitate in TA cloning.BamHI and SalI sites were ENgineered in PCR Primers.I always collect the insert(BamHI and SalI site) from the Gel and ligate with the similar pET28a sticky ends (1:5 ratio+ eq vol of Takara SolI 16 degree over night incubation).
If my insert was not there in the Expression vector, I would not have got PCR amplification of my insert using specific primer.But double and single digestion showing same results making me confused!!!!
1. Try restreaking your construct. It sounds as if there might be a mixed population of cells.
2. Cut and run on the same gel some of the pET28a plasmid. This will give you a standard to compare the size of your plasmid with the one which allegedly has your GOI.
3. It would be helpful to see the gel, and hear the sizes of the bands you expect to see.