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#1 Qiaochu

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Posted 18 August 2014 - 09:41 PM

Hi all,

I am a starter for cloning. I need help. Thanks

 

Purpose: Insert the plasmid (1230bp) into vector (6650) by Ecor1 and Xho1 REs

 

Procedure: 1. PCR (primer design with ECOR1 and Xho1 at the end and another 6 bp for protecting the RE site)

                   2. Run the DNA gel, see clear single bands, and gel recovery under UV light

                   3. Digest the PCR product and vector with ECOR1 and Xho1 together since the same buffer system for both REs. 4h 37 oC. 65C                                    deactivate. Then add CIP for both vector and PCR product 37C 30 min.

                   4. Run DNA gel, see clear bands, then do gel recover under UV light.

                   5. Ligation: T4 ligase (invitrogen) 16 C overnight, ratio between insert and vector was 3:1, 6:1, 9:1.

                   6.Transformation: DH5alph competent cells, heat shock 42 C, 90 sec. agar plate with carbenicillin. (resistance amp)

 

Results: 1) set up positive group, transformation the vector without REs (circular vector), get colonies

              2) set up a gourp, which only cut the vector with one RES either ECOR1 or Xho1, then ligation. After transformation, do not get any                            colonies

              3) others ligation product do not get any colonies. 

 

Problem: 1. UV harm the DNA?

               2. 5x ligation buffer (invitrogen) has PEG, is it ok for ligation system?

               3. Do I need do phenol-chloroform participation instead of gel recovery?

 

How fix it?

                



#2 pito

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Posted 18 August 2014 - 10:51 PM

I have not checked it in detail, but what I noticed right away is that you both treat the vector and PCR product with the CIP?

 

Hi all,

I am a starter for cloning. I need help. Thanks

 

Purpose: Insert the plasmid (1230bp) into vector (6650) by Ecor1 and Xho1 REs

 

Procedure: 1. PCR (primer design with ECOR1 and Xho1 at the end and another 6 bp for protecting the RE site)

                   2. Run the DNA gel, see clear single bands, and gel recovery under UV light

                   3. Digest the PCR product and vector with ECOR1 and Xho1 together since the same buffer system for both REs. 4h 37 oC. 65C                                    deactivate. Then add CIP for both vector and PCR product 37C 30 min.

                   4. Run DNA gel, see clear bands, then do gel recover under UV light.

                   5. Ligation: T4 ligase (invitrogen) 16 C overnight, ratio between insert and vector was 3:1, 6:1, 9:1.

                   6.Transformation: DH5alph competent cells, heat shock 42 C, 90 sec. agar plate with carbenicillin. (resistance amp)

 

Results: 1) set up positive group, transformation the vector without REs (circular vector), get colonies

              2) set up a gourp, which only cut the vector with one RES either ECOR1 or Xho1, then ligation. After transformation, do not get any                            colonies

              3) others ligation product do not get any colonies. 

 

Problem: 1. UV harm the DNA?

               2. 5x ligation buffer (invitrogen) has PEG, is it ok for ligation system?

               3. Do I need do phenol-chloroform participation instead of gel recovery?

 

How fix it?

                

 


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#3 bob1

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Posted 19 August 2014 - 02:08 AM

If you have single bands from a PCR, there is no need to gel extract, at the most you should clean them up by precipitation or PCR purification kit. The same goes for your vector, if the bit you are cutting out is smaller than about 20-30 bp, then you don't need to gel extract, just clean up by one of the above methods.

 

Avoid phosphatases whereever possible, they cause more trouble than they are worth (especially CIP), as you have incompatible ends from your two different restriction enzymes, you don't need to dephosphorylate. You never need to dephosphorylate the insert.

 

Your ligation buffer should be fine, just make sure it is 1x in the ligation reaction.

 

ligations work best when  you have 20 ng or less of vector.  Make sure your ratios are molar ratios not amount ratios!



#4 phage434

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Posted 19 August 2014 - 04:06 AM

Skip both gel cleanups. You need to clean up the pcr product (but not the vector digestion or pcr insert digestion) to remove the pcr enzyme and  dntps, but you should do this with a column. The 65C temperature at the end of your RE digestions inactivates the REs. You should be able to go directly from that inactivated RE digestion into a ligation. I would recommend against a PEG containing ligation buffer here, since you have cohesive ends. Use the normal ligation buffer, and as Bob1 says, don't use too much DNA.

A common problem is poor competence cells. You can test for this by transforming serial 10x dilutions of your parent plasmid and claculating the efficiency, normally expressed as CFU/ug of vector. Ideally you should get around 10**8 colonies per microgram of vector.

If things are working well, you need only about 15 minutes at room temperature for ligation. Overnight for cohesive end ligations is simply wasting your time.



#5 Qiaochu

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Posted 19 August 2014 - 05:44 AM

I have not checked it in detail, but what I noticed right away is that you both treat the vector and PCR product with the CIP?

 

Hi all,

I am a starter for cloning. I need help. Thanks

 

Purpose: Insert the plasmid (1230bp) into vector (6650) by Ecor1 and Xho1 REs

 

Procedure: 1. PCR (primer design with ECOR1 and Xho1 at the end and another 6 bp for protecting the RE site)

                   2. Run the DNA gel, see clear single bands, and gel recovery under UV light

                   3. Digest the PCR product and vector with ECOR1 and Xho1 together since the same buffer system for both REs. 4h 37 oC. 65C                                    deactivate. Then add CIP for both vector and PCR product 37C 30 min.

                   4. Run DNA gel, see clear bands, then do gel recover under UV light.

                   5. Ligation: T4 ligase (invitrogen) 16 C overnight, ratio between insert and vector was 3:1, 6:1, 9:1.

                   6.Transformation: DH5alph competent cells, heat shock 42 C, 90 sec. agar plate with carbenicillin. (resistance amp)

 

Results: 1) set up positive group, transformation the vector without REs (circular vector), get colonies

              2) set up a gourp, which only cut the vector with one RES either ECOR1 or Xho1, then ligation. After transformation, do not get any                            colonies

              3) others ligation product do not get any colonies. 

 

Problem: 1. UV harm the DNA?

               2. 5x ligation buffer (invitrogen) has PEG, is it ok for ligation system?

               3. Do I need do phenol-chloroform participation instead of gel recovery?

 

How fix it?

                

 

Yes. correct. Do I need remove this step?

 

Qiaochu



#6 Qiaochu

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Posted 19 August 2014 - 05:49 AM

If you have single bands from a PCR, there is no need to gel extract, at the most you should clean them up by precipitation or PCR purification kit. The same goes for your vector, if the bit you are cutting out is smaller than about 20-30 bp, then you don't need to gel extract, just clean up by one of the above methods.

 

Avoid phosphatases whereever possible, they cause more trouble than they are worth (especially CIP), as you have incompatible ends from your two different restriction enzymes, you don't need to dephosphorylate. You never need to dephosphorylate the insert.

 

Your ligation buffer should be fine, just make sure it is 1x in the ligation reaction.

 

ligations work best when  you have 20 ng or less of vector.  Make sure your ratios are molar ratios not amount ratios!

Thanks. I will avoid the gel extraction because my cutting out was pretty small. In my ligation system, the total DNA was 100 ng, which means the sum of vector and insert.. Is that too low?

 

One question, if I choose the precipitation procedure, I should choose phenol-chloroform or chloroform-enthanol procedure? which one is better? Because I have to prepare these solution by homemade.

 

Qiaochu



#7 Qiaochu

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Posted 19 August 2014 - 05:54 AM

Skip both gel cleanups. You need to clean up the pcr product (but not the vector digestion or pcr insert digestion) to remove the pcr enzyme and  dntps, but you should do this with a column. The 65C temperature at the end of your RE digestions inactivates the REs. You should be able to go directly from that inactivated RE digestion into a ligation. I would recommend against a PEG containing ligation buffer here, since you have cohesive ends. Use the normal ligation buffer, and as Bob1 says, don't use too much DNA.

A common problem is poor competence cells. You can test for this by transforming serial 10x dilutions of your parent plasmid and claculating the efficiency, normally expressed as CFU/ug of vector. Ideally you should get around 10**8 colonies per microgram of vector.

If things are working well, you need only about 15 minutes at room temperature for ligation. Overnight for cohesive end ligations is simply wasting your time.

Thanks for your suggestion,.

 After the REs digestion for both insert and vector, do I need precipitate them by phenol-chloroform or chloroform-enthanol procedure. then do the ligation? How about remove some vector or insert which do not digest completely?

 

Qiaochu



#8 pito

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Posted 19 August 2014 - 11:36 AM

If you dephosphorylate both of them , it wont ligate..

 

You can use an ethanol precipitation, but a PCR clean up is good too.

 

Most of it should be digested, part of insert that is not digested is not such a big problem. The vectoir that is not digested: you can cut with a 3the RE prior to the transformation. Take a RE that cuts in the site you have removed. Vectors that were still complete, will be cut by this enzyme and the digested (+insert ligated) will not be cut.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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