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Ligation and Transformation help needed: pENTR1A Vector issue


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#1 CambridgeBiochemist

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Posted 01 January 2010 - 05:46 AM

Dear all,

I have been looking around this site and it seems many people are getting their problems solved..so I will try my luck.

Currently, I am trying to insert my gene (1800bp) into pENTR1A vector.
Gene to replace the lethal ccd gene in pENTR1A

Steps done:
My gene was PCR amplified from pGEX6p-2 vector
--> Agarose gel run (1800bp band seen)
--> PCR reaction purified using Quiagen kit
--> Gene and vector (pENTR1A) digested with SalI and NotI
(FastDigest Enzymes used), ~1hour incubation, 37C, Insert: 50uL reaction (enzyme used: 5/50=10%), Vector: 20uL reaction (enzyme used: 2/20=10%)
---> Agarose gel run -> insert digestion: ~1800bp band seen (expected); vector digestion: ~ 2200bp band seen (as expected) --> both gel purified using Quiagen kit
--> Ligation (Fermantas T4 DNA Ligase) 20uL reaction, 2/20: 10% Ligase used, 1hour, 22C, 1:3 (vector: insert)
--> Transformation 1: 6uL of Ligated DNA into 100uL dh5alpha cells (standard protocol)
--> Gel Run -> 4000bp ligated band seen (though faint) --> gel Purified
--> Transformation 2: 6uL of Gel purified Ligated DNA into 100uL dh5alpha cells

Transformation results:
Transformation 1: One colony
Transformation 2: No colony (surprising since I have pure ligated product, but little agarose might have inhibited transformation?)

Next Steps:
Transformation 1: One colony -> 5mL Overnight Culture, 40ug/mL KAN, 37C 300rpm -> Miniprep ->
-> Absorbance check: 6ng/uL reading
-> Gel check -> No band found

query: How come no plasmid, if I got the colony?
-Undigested pENTR1A vector into dh5aplha cells should not grow due to lethal ccd gene
-Partly digested pENTR1A cannot go inside the cells and pENTR1A cannot ligat to itself since it was cut with two different enzymes..

So how did I get that colony w/o ligated pENTRA1A vector in it?

Next: I doubted I got low plasmid, so was not able to detect it...I did Maxi Prep..(150mL culture)
--> Whole of Maxi prep final solution loaded onto gel -> no band
For sure I don't have ligated pENTR1A in that colony..

Perhaps, undigested pENTR1A got in and that colony is resistant to lethal ccd gene.
or more likely, that colony of dh5alpa is resitant to KAN so it grew..!!

So at the end of the day, I have not got my ligated pENTR1A vector into dh5alpha cells:

That is not yet all: I did transformation controls
(1) dh5cells spreaded on LB plate (no antibiotic) -> many colonies -> dh5 cells are alive
(2) pENTR1A transformed into dh5alpha spreaded on LB KAN plate -> no colonies (as expected, ccd gene in pENTR1A is lethal)
(3) KAN LB plate w/o cells incubated -> no colonies -> so no background colonies
(4) dh5cells spreaded on LB KAN plate -> no colonies -> Kanamycin is working
So the dh5alpha cells and Kanamycin is working alright. But I must admit, I can't say that transformation is working alright since undigested pENTR1A is lethal to cells and as expected I don't get colonies...now that is due to ccd killing cells or KAN killing dh5cells w/o pENTR1A...it's difficult to tell...

However, I did a transformation a month before with a pGEX6P2 vector into dh5alpha and got colonies, so I feel transformation is working..
Taking that forward...that transformation is working....it's something to do with ligation...

I repeated ligation: this time overnight ligation: 4C, 18hours, 1:3 (V:I), 20uL reaction, 5% ligase -> Did two transformations:
T1: 1uL into 50uL cells -> no colonies
T2: 3uL into 50uL cells -> no colonies

So still ligation trouble exists...any suggestions?...You know I have been really working day and night with exp and controls...so I need an expert advice..Thanks in advance..

#2 phage434

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Posted 01 January 2010 - 07:19 AM

First, let me congratulate you on the fine detail provided in your post. It's great to have a question asked so carefully and with the controls spelled out.

Two problems seem possible. First, SalI is a known troublemaker in digesting pcr fragments. I'm not sure the reasons are known, but make sure you have sufficient 5' extensions on your pcr primers. If I had no other choice for an enzyme, I might try a phenol/chloroform extraction and ethanol precipitation prior to ligation. NEB has a high fidelity version of SalI available. They document its poor ability to cut pcr fragments. It is fussy in its buffer requirements and is inhibited by the presence of nucleotides. They say it cuts with only 3 bp of 5' overhang, but I would definitely use 8-10.

You didn't mention the amounts of DNA that you were ligating. Remarkably, less is often better. I aim for 20 ng of vector total in the ligation mix and equimolar amounts of insert, though more insert is sometimes recommended. This is because high concentrations of vector and insert encourages concatamers rather than recircularization. For longer fragments, use lower concentrations (happens automatically if you use constant weight, but you want even less than would be implied by that, since is it linear length vs. volume accessible to the ends).

Secondly, I would definitely check my transformation efficiency. Use any plasmid that will transform your cells and serially dilute to 10 pg/ul. Transform your cells with 1 ul (10 pg), and plate all of the cells. You should get hundreds or thousands of colonies (1000 = 10^8 transformants/ug, a decent efficiency). How are your competent cells prepared or purchased?

#3 CambridgeBiochemist

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Posted 03 January 2010 - 03:06 PM

Thank you for your comments.

I was not aware that SalI is a troublemaker. There are 4 nucleotides before SalI restriction sequence in the PCR product. SalI could be a problem and also it's difficult to resolve the undigested and SalI only digested PCR product on gel.

A positive transformation CTRL was performed:
50ng pENTR-GUS plasmid (ccd lethal gene replaced by GUS) transformed into 50uL dh5alpha and latter spread on LB KAN plate, 37C, 16hrs -> Colonies seen. This indicates transformation is working.

It's either to do with ligation or digestion. To me SalI digestion seems to be the suspect. So, first I will eliminate NotI involvement by performing digestion controls with NotI only digestion with both pENTR1A and PCR product (NotI will cut out 20ish bp from PCR product, so it might be possible to resolve the NotI only and undigested PCR product. If NotI is working then that leaves SalI being the primer suspect, then I will stop all further investigation and order new primers with different restriction site.

Regarding dh5 alpha cells: They are lab prepared and they work fine for everyone in the lab. I have not done the serial dilutions to check it's transformation efficiency, but since myself have used these cells earlier with similar DNA transformation concentrations used now and it worked fine, so its safe to assume that transformation is not a problem.

Coming back to Ligation question: I have been using (ng) 100:300 (V:I) for all ligations. However, taking your advice I performed three more ligations:

Ligation 1, NEB T4 DNA Ligase (3.3%), 1:1 (20ng:20ng) (V:I) (Total <10%), 30uL Reaction, 22C, 1.5hrs -> Transformation -> No colony
Ligation 2, NEB T4 DNA Ligase (3.3%), 1:3 (20ng:60ng) (V:I) (Total <10%), 30uL Reaction, 22C, 1.5hrs -> Transformation -> No colony
Ligation 3, NEB T4 DNA Ligase (3.3%), 1:5 (20ng:100ng) (V:I) (Total <10%), 30uL Reaction, 22C, 1.5hrs -> Transformation -> No colony

So, back to the original problem: SalI might be problem, I will be back after NotI digestion CTRLs.

Mean time all suggestions are wholeheartedly welcome. Thanks in advance.


First, let me congratulate you on the fine detail provided in your post. It's great to have a question asked so carefully and with the controls spelled out.

Two problems seem possible. First, SalI is a known troublemaker in digesting pcr fragments. I'm not sure the reasons are known, but make sure you have sufficient 5' extensions on your pcr primers. If I had no other choice for an enzyme, I might try a phenol/chloroform extraction and ethanol precipitation prior to ligation. NEB has a high fidelity version of SalI available. They document its poor ability to cut pcr fragments. It is fussy in its buffer requirements and is inhibited by the presence of nucleotides. They say it cuts with only 3 bp of 5' overhang, but I would definitely use 8-10.

You didn't mention the amounts of DNA that you were ligating. Remarkably, less is often better. I aim for 20 ng of vector total in the ligation mix and equimolar amounts of insert, though more insert is sometimes recommended. This is because high concentrations of vector and insert encourages concatamers rather than recircularization. For longer fragments, use lower concentrations (happens automatically if you use constant weight, but you want even less than would be implied by that, since is it linear length vs. volume accessible to the ends).

Secondly, I would definitely check my transformation efficiency. Use any plasmid that will transform your cells and serially dilute to 10 pg/ul. Transform your cells with 1 ul (10 pg), and plate all of the cells. You should get hundreds or thousands of colonies (1000 = 10^8 transformants/ug, a decent efficiency). How are your competent cells prepared or purchased?



#4 phage434

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Posted 03 January 2010 - 04:39 PM

Note that your control is transforming 50,000 times as much plasmid as the control I suggested. Your efficiency may not be adequate. You should get a lawn of 50 million colonies if you had 10^8 cfu/ug efficiency.

The fact that you are seeing zero colonies is troublesome. Usually, even for failed ligations, you see a few colonies, just not the ones containing your insert. I know the plasmid is supposed to contain ccdB, but I have found that you still see some background. That you are seeing none indicates to me that the problem may be transformation efficiency.k

I would immediately order new primers with a different restriction site, if this is an option. The cost is small compared to your time and effort.

#5 hanming86

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Posted 03 January 2010 - 07:08 PM

Gel extraction typically causes quite a lot of downstream problems. I would recommend doing an ethanol precipitation on the eluted sample to rid of any remaining agarose or other inhibitor.

However in your case, i think you might be able to eliminate gel extraction if i m analyzing ur information right. i did make an assumption that the distance between NotI and SaiI cutsite is quite small <50bp perhaps.

Using this assumption here..

What i would do.

AFter u PCR out the gene using primer which will give another more bases before the SaiI site


1. digest vector/ pcr fragment separately
2. NotI and SaiI could be run in the same buffer, both are heat inactivatable, that's good.
3. after digestion, heat inactivate, ethanol precipitation
4. ligation
5. transform

regards,

Ming
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#6 CambridgeBiochemist

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Posted 04 January 2010 - 02:44 AM

Hi Ming,

The distance between SalI and NotI sites in the

PCR product: ~ 1900bp
pENTR1A: ~450bp

After digestion, I have purified using PCR purification kit (Quiagen) rather gel extraction the second time, so agarose should not be the problem.

I take the previous point to ethanol precipitate the ligated DNA and then perform transformation.


Gel extraction typically causes quite a lot of downstream problems. I would recommend doing an ethanol precipitation on the eluted sample to rid of any remaining agarose or other inhibitor.

However in your case, i think you might be able to eliminate gel extraction if i m analyzing ur information right. i did make an assumption that the distance between NotI and SaiI cutsite is quite small <50bp perhaps.

Using this assumption here..

What i would do.

AFter u PCR out the gene using primer which will give another more bases before the SaiI site


1. digest vector/ pcr fragment separately
2. NotI and SaiI could be run in the same buffer, both are heat inactivatable, that's good.
3. after digestion, heat inactivate, ethanol precipitation
4. ligation
5. transform

regards,

Ming






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