problem with cloning of dsred plz help - vector cut seems fine, still religation oocurs (Jun/14/2010 )
Hello
I am using a vector called SS1A1 which is not commercially available, not sequenced in our lab.
In MCS, there was SPR cloned in Xba1/ Kpn1 site, which i released and added additional restriction sites into it.
My problem now is : I was trying to clone DsRed into ECOR1 site, I digest the vector, it runs fine on the gel, seems to be lineralised.
i give SAP treatment, still i can see the colonies on the control plate after ligation?
Its kind of annoying:(
I assume that after SAP treatment, there should not be religaton of the vector?
Then how is that am getting colonies in the control plate?
Whther we need to gel extract the vector after restriction digestion with ECOR1?
I am thinking, as am directly adding SAP buffer, buffer incompatibilty is causing SAP not to work?
Kindly suggest and help me. Its taking too long time to clone.
You should always gel-purify both your linearized vector and your insert fragment. It's much more likely you're getting vector-only colonies on your transformation plates because there is uncut vector in your transformation than it is because your SAP is not working.
HomeBrew on Jun 14 2010, 08:39 PM said:
Thank you for the reply. Am using EcoR1 site for cloning. I was worried about the star activity. I was keeping it for shorter duration. whats the maximum time limit for the star activity to start? Can u please suggest. I am using 20 U/ ul concentration of enzyme.
Star activity is usually triggered by by inappropriate digestion conditions, not by time of digestion. I have never found star activity to be much of a problem with EcoRI, especially in a single-enzyme digest.
Besides, star activity causes the enzyme to cut not only at its own recognition sequence, but also at inappropriate places. If you gel purify your cut vector, and only recover the full-length band, star activity is not an issue.
Thank u once again. I am now doing the way u suggested. Hope it goes fine this time.
I did the way u suggested me, I gel extracted both vector and insert and then gave SAP treatment for vector.
I can see more colonies on control plates than on transformed plates.
I picked some colonies to screen, but am not sure how it goes as there are colonies on controls too..
Please suggest me why is that am getting colonies on control plates even after SAP treatment?
Your antibiotics expired? Wrong antibiotics? Contaminates E.coli strains (happened to me before)?
I think to make meaningful suggestions, we need more information about what you did and how you did it. Can you give us more detail? How you did the digestion, how you did the SAP dephosphorylation, the gel purification, the ligation, the transformation, what antibiotic you're using, etc.
For example, when you say you saw "more colonies on control plates than on transformed plates", how many colonies did you see on the plates? If you saw, say, 15 colonies on the control plate and 10 on the experimental plates, you're not really seeing "more", you're seeing the same amount, within pipetting error, etc. If, however, you're seeing 100 colonies on the control plate, and 5 on the experimental plates, then you really are seeing more colonies -- details like these will help us help you figure out where the difficulty lies.
HomeBrew on Jun 18 2010, 08:01 PM said:
For example, when you say you saw "more colonies on control plates than on transformed plates", how many colonies did you see on the plates? If you saw, say, 15 colonies on the control plate and 10 on the experimental plates, you're not really seeing "more", you're seeing the same amount, within pipetting error, etc. If, however, you're seeing 100 colonies on the control plate, and 5 on the experimental plates, then you really are seeing more colonies -- details like these will help us help you figure out where the difficulty lies.
shreelatha on Jun 22 2010, 10:16 AM said:
HomeBrew on Jun 18 2010, 08:01 PM said:
For example, when you say you saw "more colonies on control plates than on transformed plates", how many colonies did you see on the plates? If you saw, say, 15 colonies on the control plate and 10 on the experimental plates, you're not really seeing "more", you're seeing the same amount, within pipetting error, etc. If, however, you're seeing 100 colonies on the control plate, and 5 on the experimental plates, then you really are seeing more colonies -- details like these will help us help you figure out where the difficulty lies.
The antibiotic used was ampicillin. I gave ecor1 digestion for 7 hours, NEB , as there is no star activity till 8 hours or more.
I then heat inactivated vector at 65 degree / 20 mins. I made clean up of vector after heat inactivation.
Insert was cloned at in pGEMT, so i gel extracted it after treating with ecor1 for the same interval as the vector. I loaded both insert and vector on the gel after gel extraction and clean up, I could see nice linear band on loading 1 ul each of them.
After which i gave SAP treatment of the vector, for 45 mins, heat inactivated it for 15 mins @ 65 degree. SAP used was from enzynomics.
I calucated the molar ratio of 1:3 and 1:5 and added vector and insert accordingly. Set up liagtion at 18 degree over night and then transformed.
After transformation, (heat shock) I could see ~80 colonies on control plate ( vector alone) and ~ 100 colonies or same on both 1:3 and 1:5 plate.
:( I screened few colonies, still all were reliagted ones. I have done this many many times now, i am really wondering why its not working?
Please help.