Hi Miriam

"Are these values normal? too high?"

It depends on the amount of PCR product you have and the volume you used for elution. But if your nanodrop is calibrated, they should be ok.

did you purified your PCR product after adding A?

"I calculated from promega 1:3 ratio of my insert (0.5 kb) and the pGEM vector"

If you want to do a molar ratio of 3 insert and 1 vector, and using 25 ng of pGEM-T, you should add 12.5 ng of insert, because your insert is 6 times shorter than vector, so 25/6=4.17 ng x 3 (to get the 3:1 ratio insert:vector) = 12.5 ng. I usually use 50 ng of pGEM-T.

"Obtained a huge number of white colonies in both + control". so, your competent cells and buffer are ok.

"my ligation reactions, isolated plasmid DNA and digested with RE non showed a band on the gel".

This means you got colonies on the plate? How many colonies? Sometimes you get 5-20 colonies on negative control (ligation reaction with pGEM-T and no insert).

My advice:

-always purify your PCR product after any enzymatic reaction.

-do the calculations again to get the ratio you really want.

-try a higher ratio, 6 insert:1 vector, for example.

-do always a negative control (ligation with pGEM-T w/o insert).

-try to do the ligation a 4º degrees overnight.

-make sure your A overhanging is working properly.

Hi, if I'm understanding correctly you have tried to add an A tail to a PCR product and want to ligate it into a TA type pGem vector?

If so, I would just repeat PCR with a taq enzyme that automatically adds an A tail to the product for easy cloning. There are a multitude of these and especially since you are cloning using a promega kit, I would suggest using GoTaq. If you are concerned with fidelity of the product there are many hi fidelity type taq mixes that also add an A tail.

If you are still set on adding an A tail then lighting, I would make sure you purify your PCR after adding the A tail, and re nanodrop your sample (you should never calculate DNA concentration on a dirty PCR). Then I would set it up as follows for the first product:

Since you have 227ng/ul (pretty good PCR yield) and you need will add 25ng of backbone, this means you should calculate it in terms of pmol ends -> your backbone is 3kb but your insert is only 0.5kb therefore to make sure you have a 3:1 ratio of pmol ends you need to calculate your insert concentration to 1.5kb.

(1.5 x 25)/3 = 12.5ng of insert needed.

Since you have 227ng/ul you only need to add 0.055ul of your product.

My advice would be to dilute the product and use that for your ligation.

E.g.

1ul of PCR product + 9ul of H2O (1/10 dilution) = 10ul of PCR product at 22.7ng/ul

And therefore you would use 1.8ul of this in your ligation.

Finally regarding your colonies, the fact that you are getting so many white colonies on your negative plate is worrying. You should not be getting many white colonies on your negative plate, make sure your pipettes are clean, your reagents are contaminant free and that you prepare your negative control (ligation without any insert) before you do the one with the insert.

When you finally do get white colonies on your ligation plate (and not many in your negative) make sure you digest screen and determine the orientation of your product in the vector. The product can insert either way into the vector so make sure to pick RE sites that will give you two distinct bands one corresponding to your backbone 3kb and one for your insert 0.5kb (you can even do a triple digest if you are wanting to be sure).

And lastly once you have obtained DNA from a few colonies which all located correctly, you need to sequence the insert to be sure that there were no errors in the PCR.

If you try all of this and still have trouble pls let me know exactly what you are doing and what the problems are and I will try to help more,

Good luck :)

-try a higher ratio, 6 insert:1 vector, example.

NO THIS WRONG, do not increase volume of insert (PCR product have inhibitors for the ligation process) ........ you should use 2ul or maximum 3ul, also your volume 1 ul is ok ......

-make sure your A overhanging is working properly.

YOU SHOULD USE FRESH PCR PRODUCT before the overhang is affected.

-try to do the ligation a 4º degrees overnight.

I did it at 14c (not 4 c) overnight and it worked.

1. pcr = 227 ng/ul (500 bp)

2. pcr = 494 ng/ul (500 bp)

(This DNA conc is so high for 500 bp purified PCR product ... it should be around 50 ng / ul !!! ....

You should increase vector to 2 ul and PCR product to 2 ul and 1ul enzyme is enough (AVOID FREEZING AND THAWING OF LIGASE, ALSO AVOID LEAVING IT IN ICE FOR LONG PERIOD, PUT YOUR VOLUME AND GO RIGHT AWAY TO FREEZER) .....

@Ibrahim:

"-try a higher ratio, 6 insert:1 vector, example.

NO THIS WRONG, do not increase volume of insert (PCR product have inhibitors for the ligation process) "

NO, IT´S NOT WRONG. I did many times (hundreds) ligations into pGEM-T with 0.2, 0.5, 1, 1,5 or 2 kb inserts. They always worked fine. I was talking about purified PCR products (read the advice I gave to Miriam) and quantified by comparison with DNA ladder in agarose gel.

"-try to do the ligation a 4º degrees overnight."That´s right.

"You should increase vector to 2 ul and PCR product to 2 ul" But what is the concentration of both? In any experiment you design, it should be better to work with concentrations, instead of volumes... 2 uL of vector means 100 ng, what it´s a waste of vector. Much better to use a fixed amount of vector (50 ng, 1 uL) and calculate the molar ratio you want for your insert (as the manual recommends)

By the way, you don´t freeze and thaw the ligase, it´s in 50% glycerol. Keeping the ligase vial in a stratacooler when using is enough.

@ José Angel Campos-Sando

Sorry, maybe my words were tough ... please accept my apologize.

But I did face troubleshooting at UMN before with T/A cloning while using reverse genetics for influenza A virus that's why I go back to invitrogen protocols ... they adviced me by this ...... not to increase conc. of PCR product because of inhibitors .... the same for ligation degree (14 c) overnight, other kits was in room temperature for 5-10 minutes for rapid ligation .... ... when I changed my protocol I did get better results ...

https://www.lifetechnologies.com/eg/en/home/references/protocols/cloning/ligation-protocol/cloning-of-a-tailed-pcr-fragments-using-conventional-ligase-method.html

TROUBLSHOOTING:-

Incorrect molar ratio of vector:insert used in the ligation reaction.

(Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio). so no 1:6 ratio

The PCR products were stored for a long period of time before performing the ligation reaction.

(Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage).

It seems the we used kits from different companies which have different approaches ..... sorry about the conflict .... it is the same principle but different companies ...

Mariam check the attached file for troubleshooting ...

@Ibrahim

1- When you use capitals to reply, it´s like you´re shouting, what it´s not correct here.

2-There are many protocols, ratios, kits... I tested many and chose one that works fine for me, but don´t say other people they are wrong, because they can be more experienced than you.

3-Maybe I´m wasting time in the lab doing this, but I always try to estimate the concentration of my purified products and do my calculations using concentrations, because I like to know what I´m going to add to the reaction. Volume don´t say too much: in the same volume, you could have 1 ng of PCR product or 100 ng.

But no problem. It´s a normal discussion between colleagues.

Best regards

Ok, Sure, it is a good discussion and useful for all of us, the case is only that you do not want other people to spend a lot of effort as you did before to solve the problem that's why I did answer to Mariam according to knowledge as much as I can.

For volumes and concentrations I usually do not quantify every sample I do have because sometimes you have valuable samples with limited amount, so in every time you quantify you lose 2 ul of your sample, what would be the case when you repeat the experiment 3 times in each time you lose 2 ul and if you need this sample for other experiments .... in that case I was following the attached file in order to quantify every time and waste time and samples ..... but sure you are right quantification is better ....

Different points of views in science is every useful.

Best Regards.

@ José Angel Campos-Sando

When you use capitals to reply, it´s like you´re shouting, what it´s not correct here.

There are many protocols, ratios, kits... I tested many and chose one that works fine for me, but don't say other people they are wrong, because they can be more experienced than you

((Yup!), you are right and I can understand this).

Taq polymerase adds A overhangs on pcr products so you dont need an extra reaction if using Taq, gel purification of pcr product is another good option, molar end calculations are very important for conventional cloning as it matters a lot as far as incubation time is concerned 2 hour incubation works well normally. avoid using too much pcr product as well as vector as it leads to complexities and false results.

It is better to use less concentrated PCR Products and Vector because large concentration result complexity and wrong interpretation.

i have amplified a pcr product of about 1.1kb and gene clean it but for after ligation (Ptg-19-T) icubation at 22 C for one hour and than at 16 C overnight and cloned them in DH5 alpha cells. i have also used a plasmid as control. i get abundant blue colonies in control but not even a single colony in amplified product plate. Kindly help me in this regard.