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pcDNA 3.1 Directional TOPO clone issues

ligation transformation

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18 replies to this topic

#1 Jeremy55

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Posted 01 December 2013 - 07:03 PM

Hello, everyone

I'm fresh here. And I have some problems with pcDNA 3.1 Directional TOPO cloning. I've been trying to insert a 3KB fragment into the pcDNA 3.1 D/V5-His-TOPO vector,then transform the recombinant plasmid into TOP10 competent cells. However, there are no or very few colonies on the plate.

I need all of you experts' instructions. Help me please.



#2 bob1

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Posted 01 December 2013 - 07:28 PM

Welcome

 

Details would help - what have you done, how did you prepare the insert?



#3 ostiaziocan

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Posted 01 December 2013 - 09:00 PM

Welcome,

 

are those few colonies containing the insert you are expecting?



#4 Jeremy55

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Posted 03 December 2013 - 04:36 PM

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.


Edited by Jeremy55, 03 December 2013 - 04:44 PM.


#5 Jeremy55

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Posted 03 December 2013 - 04:41 PM

Welcome,

 

are those few colonies containing the insert you are expecting?

Thank you.

It's a small probability event. I need lots of clones to do my subject, and the vector is very expensive. 


Edited by Jeremy55, 03 December 2013 - 04:41 PM.


#6 bob1

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Posted 03 December 2013 - 05:02 PM

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?



#7 Jeremy55

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Posted 03 December 2013 - 05:11 PM

 

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?

 

PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.



#8 Jeremy55

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Posted 03 December 2013 - 05:19 PM

 

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?

 

Have you used the vector?



#9 bob1

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Posted 04 December 2013 - 03:02 AM


PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.

 

That should be fine.  Are you sure you added the required bases to the 5' end of the primer?



#10 Jeremy55

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Posted 04 December 2013 - 03:31 AM

 


PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.

 

That should be fine.  Are you sure you added the required bases to the 5' end of the primer?

 

Yes, I'm sure. 



#11 bob1

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Posted 05 December 2013 - 01:00 PM

How much of the ligation mix are you transforming?  Did you heat kill the ligation?  Large inserts are relatively hard to ligate, what conditions did you try (molar ratios, temperatures, amounts of insert and vector)?



#12 Jeremy55

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Posted 12 December 2013 - 12:02 AM

How much of the ligation mix are you transforming?  Did you heat kill the ligation?  Large inserts are relatively hard to ligate, what conditions did you try (molar ratios, temperatures, amounts of insert and vector)?

I've tried 2,3 and 6 μl ligation mix for transformation. And now there are more colonies on plate than before.However, most of them are false positive colonies. Plasmid minipreps from those colonies are suffered from restriction enzyme cutting (BamH I and Not I ,Fermentas) . Only few dropped targeted fragment; a few dropped very weak targeted fragment ; others just turned out the vector's size and nothing dropped away. And I still don't know why.

 

Do you mean terminating the ligation reaction by heat kill? If it is, I didn't.

 

 

At the beginning, I tried 15ng:1μl ratio of PCR:TOPO vector.But that didn't work .So, now I adopt 1μl:1μl ratio of PCR: TOPO vector (IMaybe too much false positive colonies has something with this, doesn't it? ). As to temperature, 37 degrees centigrade are the best according to the growth of colonies on plate.(I've tried room temperature and 30 degrees centigrade .Now it is in winter here ;so I abandoned room temperature conditions.)



#13 Jeremy55

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Posted 12 December 2013 - 12:12 AM

How much of the ligation mix are you transforming?  Did you heat kill the ligation?  Large inserts are relatively hard to ligate, what conditions did you try (molar ratios, temperatures, amounts of insert and vector)?

I'm a Chinese.And I'm not very good at English, especially oral English or conversation English. If there are impolite words or something else, forgive me and believe me I didn't mean that please.laugh.png



#14 bob1

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Posted 12 December 2013 - 12:02 PM

Your english is fine - we see much worse on here, even from people who are probably native speakers.

 

You need to make sure that the ligation mix is less than 5% of the total volume of your transformation, so if you had 50 ul of bacteria, you would want to use less than 5 ul of ligation.

 

Those clones that have the insert - what's wrong with using those ones?  If you pick, make glycerol stocks, and screen for the insert, then you should find the right one reasonably frequently (I usually pick about 20 colonies, will have 15 or so of those have an insert and sequence 3-5, and find the correct insert in most).   The weak target fragment is probably due to inhibitors in the digest, this is most commonly caused by not getting rid of all the medium before lysing the cells for plasmid extraction.

 

The handbook for the kit should tell you how much PCR to use with how much TOPO vector.  Using at 1:1 is not usually a good idea.  Ideally you would know the concentration of both insert (PCR) and the vector, from this you can calculate the amount of insert DNA to use by the  molar ratios using the following formula:

 

ng insert to use = ratio x (insert length (bp)/vector length (bp)) x ng of vector used

 

You want to keep the total amount of vector to less than 20 ng if possible, usually they work best with less DNA.  Ligations can be carried out at 4, 16 or room temp (22 C is standard).  The TOPO manual should tell you which one to use, but it may pay to try others.  Usually the low temp ones are done overnight.

 

Note that all these instructions apply to standard cloning, but should also be applicable to TOPO cloning.



#15 Jeremy55

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Posted 14 December 2013 - 01:08 AM

Your english is fine - we see much worse on here, even from people who are probably native speakers.

 

You need to make sure that the ligation mix is less than 5% of the total volume of your transformation, so if you had 50 ul of bacteria, you would want to use less than 5 ul of ligation.

 

Those clones that have the insert - what's wrong with using those ones?  If you pick, make glycerol stocks, and screen for the insert, then you should find the right one reasonably frequently (I usually pick about 20 colonies, will have 15 or so of those have an insert and sequence 3-5, and find the correct insert in most).   The weak target fragment is probably due to inhibitors in the digest, this is most commonly caused by not getting rid of all the medium before lysing the cells for plasmid extraction.

 

The handbook for the kit should tell you how much PCR to use with how much TOPO vector.  Using at 1:1 is not usually a good idea.  Ideally you would know the concentration of both insert (PCR) and the vector, from this you can calculate the amount of insert DNA to use by the  molar ratios using the following formula:

 

ng insert to use = ratio x (insert length (bp)/vector length (bp)) x ng of vector used

 

You want to keep the total amount of vector to less than 20 ng if possible, usually they work best with less DNA.  Ligations can be carried out at 4, 16 or room temp (22 C is standard).  The TOPO manual should tell you which one to use, but it may pay to try others.  Usually the low temp ones are done overnight.

 

Note that all these instructions apply to standard cloning, but should also be applicable to TOPO cloning.

Those clones that have the insert are sequenced and the result are right. Just very low success rate.

 

The handbook says 0.5:1 to 2:1 molar ratio of PCR product : TOPO vector  and room temperature are recommended .

 

I 've tried again acccording to your advice and the results are very depressing --no colonies on plate . But thank you all the same .You are very kind.







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