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problem with cloning of dsred plz help


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#1 balancer

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Posted 13 June 2010 - 11:05 PM

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.

#2 HomeBrew

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Posted 14 June 2010 - 03:39 AM

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.

#3 balancer

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Posted 14 June 2010 - 10:53 PM

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.


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.

#4 HomeBrew

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Posted 15 June 2010 - 02:11 AM

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.

#5 balancer

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Posted 15 June 2010 - 05:16 PM

Thank u once again. I am now doing the way u suggested. Hope it goes fine this time.

#6 balancer

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Posted 17 June 2010 - 05:13 PM

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..:P
Please suggest me why is that am getting colonies on control plates even after SAP treatment?

#7 Adrian K

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Posted 17 June 2010 - 08:25 PM

Your antibiotics expired? Wrong antibiotics? Contaminates E.coli strains (happened to me before)?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

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#8 HomeBrew

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Posted 18 June 2010 - 03:01 AM

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.

#9 balancer

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Posted 21 June 2010 - 05:16 PM

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.



#10 balancer

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Posted 21 June 2010 - 05:16 PM

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.



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.
:lol::(
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? :D
Please help.

#11 balancer

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Posted 21 June 2010 - 05:46 PM

Your antibiotics expired? Wrong antibiotics? Contaminates E.coli strains (happened to me before)?



I am using right antibiotic and its not expired.
comp cells too r freshly made and tested for any contamination.
Its all fine.

#12 tfitzwater

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Posted 22 June 2010 - 07:16 AM

1. Did your 80 colony background come from vector plus ligase (incomplete SAP) or vector minus ligase (uncut vector contamination).
2. Typically, unless you vector plus insert plate has more than twice as many colonies as your vector plus ligase, most (if not all) of the colonies you pick will be vector only.
3. I was a firm believer in gel-purification of vector and insert for many years. But then I discovered that using a Spin-X cartridge (with a rinse step afterwards) was sufficient to purify vectors that were double-digested in the multiple cloning site. The cloning site fragment goes through the Spin-X, leaving vector behind. This method does require that you have achieved 100% linearization of the plasmid.
You report 1:3 and 1:5 ratios (insert:vector or vector:insert). The size of the DNA fragments is important in this calculation. The routine 3:1 insert:vector assumes that you have a 1 kb insert into a 3 kb vector.

#13 balancer

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Posted 22 June 2010 - 07:40 PM

1. Did your 80 colony background come from vector plus ligase (incomplete SAP) or vector minus ligase (uncut vector contamination).
2. Typically, unless you vector plus insert plate has more than twice as many colonies as your vector plus ligase, most (if not all) of the colonies you pick will be vector only.
3. I was a firm believer in gel-purification of vector and insert for many years. But then I discovered that using a Spin-X cartridge (with a rinse step afterwards) was sufficient to purify vectors that were double-digested in the multiple cloning site. The cloning site fragment goes through the Spin-X, leaving vector behind. This method does require that you have achieved 100% linearization of the plasmid.
You report 1:3 and 1:5 ratios (insert:vector or vector:insert). The size of the DNA fragments is important in this calculation. The routine 3:1 insert:vector assumes that you have a 1 kb insert into a 3 kb vector.



80 colonies came from vector plus ligase. Yes ur right most of them are vector only now. Can u give me more information about Spin-X catridge?
I set up vector to insert ratio of 1:3 and 1:5. not other way around. I calculated molar ratio and then set up the ligation accordingly.

#14 balancer

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Posted 27 June 2010 - 09:14 PM

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?




@HomeBrew: i re did the way u suggested and used CIP instead of SAP, i got the clone, sequenced too..:D Thank u so much.:)




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