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Problem with subcloning genescript synthesized genes

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#1 FoTem21



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Posted 23 July 2015 - 08:04 AM



I received synthesized genes from Genescript in pUC57 vector two months ago. Since then, I've tried unsuccessfully to subclone them into pET28 expression vector. This is our protocol:

1.Amplify insert DNA using specific primers with flanking NdeI and XhoI restriction sequences

2. gel-purify insert to separate it from pUC57

3. digest product and pET28a with NdeI and XhoI

4. ligate pET28 and insert with T4ligase (22oC 1hr or 4oC overnight); 1:3, 1:1, 1:5 vector:insert ratio.

5. Transform into E.coli Top10cells

Each time I extract the plasmid and digest with NdeI and XhoI, the insert is not there.


I've tried several times including digesting directly from pUC57 (EcoRI/XhoI) and transforming into JM109 cells to no avail. We use thermoscientific fast digest enzymes in my lab which usually involve 5mins digestions. I extend this to 45mins/1hr, yet, no insert.


Pls, does anyone have any useful suggestions regarding this for me?



#2 labtastic



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Posted 23 July 2015 - 03:33 PM

Gel purify after digestion.


Also, are you digesting your pET28 with Nde/Xho?


Try it in the following order:


1. Amplify insert with NdeI/XhoI primers

2. PCR cleanup, elute in water

3. Digest PCR insert as well as empty pET28 with Nde/Xho (4 hrs at 37C)

4. Gel purify digested insert as well as digested pET28 plasmid

5. Ligate at room temp overnight (1 hr may not be enough...I do at least 2-3 hrs...and I get better efficiency if it's convenient to leave overnight)

6. Transform into Top10


One common problem in ligations is incomplete digestion of the vector (pET28 in this case). Any trace of undigested, intact plasmid will easily transform and make lots of colonies...among some colonies that have your insert. This is why I do a full 4 hrs of digestion for my vectors. If you find you have a lot of colonies with plasmid missing your insert, consider buying fresh restriction enzymes. In the mean time, do colony PCR on a couple dozen colonies to see if any one has your insert. It'll save you growing them up and miniprepping each one. 


Fresh ligase can also make a big difference in ligation efficiency. Also, always make sure your cells are sufficiently competent, but it doesn't sound like this is your problem. 

#3 Andrea Fortina

Andrea Fortina


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Posted 21 June 2016 - 07:22 PM

Gel purify after digestion.



Besides, did you run agrose gel to test whether the plasmid digested completely?

Andrea Fortina

Genius only means hard-working for all one's life.

CreativeBiomart, Recombinant protein expert



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