Hi Hafiz,

in order to obtain optimal results for your ligation you should measure the insert concentration after gel-purification and calculate the amount of insert needed for a desired insert:vector ratio (5:1 to 3:1 should work in most cases). It depends on the molecular size of both vector and insert. As an example please find the protocol for the pGEM-T easy vector system attached in the following link. Calculation is also included

https://www.promega.de/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vector-systems-protocol/

Best regards

Soner

Hello,

If you are using pGEM-T vector then prior to a-tailing you can just check the concentration and use accordingly for a-tailing so that the concentration of insert to be used comes within the volume for the ligation. So after a-tailing just take that much volume and set ligation overnight, it has always worked with me like this. I usually don't do PCR clean-up as there is the loss of DNA during the process as I use very less volume for a-tailing i.e. 10ul total reaction volume and I use Emerald master mix (Takara) for a-tailing.

But if you have sufficient DNA concentration, then you can follow the manual as suggested by Soner Öner-Sieben

Regards

Hi,

After your PCR reaction (total volume of 50 uL) with a Hi-fidelity DNA Pol was done, you can add 0.3-0.5 ul of Taq polymerase and 0.3 uL of 10 mM dATP in your reaction and return to thermocycler for more 5 min at 70 0C. When this step was finished you should put your sample tubes on ice and purify your fragments from agarose gel. Thus, you can go cloning your purified fragments in pGEM-T vector. Good luck!

Soner Öner-Sieben thank you very much i will ,

Divya Das Thanks for answer, i am using PMD-T19 vector

Maxuel Andrade Thanks for the answer, after gel purification, so there no need to measure the concentration?

Hi Hafiz Muhammad Rizwan,

you can check your concentration. Though, if you have a good amplification and lots of fragment in gel after purification just elute it in a small volume (about 20 uL) of nuclease free water and go to next step. Best wishes.

Maxuel Andrade thanks, after amplification, run a 1.5 % agarose, showing bright specific band ( loaded 25ul in a well), after that cut the gel and put 2 piece in one tube (50ul total), but after the purification, when checked the concentration, its 0.0, show no concentration, so

Hafiz Muhammad Rizwan in the last step of purification you should elute your fragment from column with 20 uL nuclease free water. Run a new agarose gel with 2 uL of purified fragment to check if it was eluted.

Maxuel Andrade thank you i will try by < Run a new agarose gel with 2 uL of purified fragment to check if it was eluted>.

if after that there have specific bands so can use directly for next Tailing,

or better to check the concentration ?

Hafiz Muhammad Rizwan as your PCR reaction is working fine then when it is done just add in this same reaction Taq Pol and dATP for tailing reaction (I mentioned it above) and let it run for 5 min at 70 0C. After that you can purify and check again if your fragment was eluted in a new gel. if your fragment was eluted you can start cloning in pGEM. Best wishes!

Maxuel Andrade thank you very much ,

I have tried before for another gene as; TA cloning by using PMD-T19 vector, >E-coli transformation (DH5@) into LB liquid medium without antibiotic for 1-2 hours, 37C, 200-250rpm shaking, > culture on LB agar plates containing Ampicyline antibiotic > selected single colonies into LB liquid medium containing Amp (4-5 hours, 200-250 rpm), did PCR with the same primers used for amplification, so now the sangon sequence results are not good, mismatching and not 100%,

as attached, I have tried many times, so the sequences results are same everytime, what should do to solve this problem,

Hafiz Muhammad Rizwan

What are the 2 attachments about? Is that your cDNA? How long is the cDNA? I saw in the first attachment saying (1 to 933), but in the first attachment it shows much less than that.

Yuan-Yeu Yau sir i did TA cloning by using PMD-T19 vector, >E-coli transformation (DH5@) into LB liquid medium without antibiotic for 1-2 hours, 37C, 200-250rpm shaking, > culture on LB agar plates containing Ampicyline antibiotic > selected single colonies into LB liquid medium containing Amp (4-5 hours, 200-250 rpm), did PCR with the same primers used for amplification, so the attached files are the sangar sequence results of nucleotide and protein, which are not good, mismatching and not 100% identity,,

What are the 2 attachments about?

sangar sequence results of nucleotide and protein after alignments with my target.

Is that your cDNA?

After TA cloning,

How long is the cDNA?

933bp

I saw in the first attachment saying (1 to 933), but in the first attachment, it shows much less than that.

For nucleotide just attached the results showing the identity and coverage %

Hafiz Muhammad Rizwan So, the 933 bp is the whole length of your cDNA, which contains a start codon at the beginning and a stop codon at the end of the sequence?

1. How did you get your cDNA? From mRNA (reverse transcripase)?

2. What did you mean by " I have tried many times, and the sequences results are same every time "? Did you mean you *sequence* the same clone a few times?

3. I saw 3 mismatches in the first attachment. Any other mis-matches in the part you did not post??

4. What DNA polymerase did you use for PCR amplification? Name of the DNA polymerase (and web link)? Is it a High Fidelity one?

Yuan-Yeu Yau

Yes sir,

1. How did you get your cDNA? From mRNA (reverse transcripase)?

Yes sir from mRNA,

2. What did you mean by " I have tried many times, and the sequences results are same every time "? Did you mean you *sequence* the same clone a few times?

Sir, after amplification> TA cloning > sequenced many times, by using different clones for every time, but the same results, mean same place mismatches.

3. I saw 3 mismatches in the first attachment. Any other mis-matches in the part you did not post??

yes, the remaining part also has 4 mismatches, I have attached the files (alignments),

4. What DNA polymerase did you use for PCR amplification? Name of the DNA polymerase (and web link)? Is it a High Fidelity one?

i am using PrimeSTAR® Max DNA Polymerase from TAKARA (https://www.takarabio.com/search-results?term=R045A),

Yes sir, it's high-fildelity.

Hafiz Muhammad Rizwan 8, this is a lot. The total 8 mismatches cause how many amino acid sequence to change? Do you think the reference sequence you used to align your query sequence is perfect? I meant does it reflect the true gene sequence of the original gene in the genome? Had the *reference sequence* been used to do any experiment in a publication and worked?

Yuan-Yeu Yau

5 amino acid sequence change,

Do you think the reference sequence you use to align your query sequence is perfect? I mean it reflects the true gene sequence of the original gene in the genome?

The reference sequence I am using for alignments is selected after BLAST with Arabidopsis from my RNA seq data,

Hafiz Muhammad Rizwan

1. Had the *reference sequence* been used to do any experiment in a publication and worked? (reference of the paper?)

2. According to my knowledge, you are using passion fruits in your research. So, did this 'total RNA' you isolated to make your cDNA is from Arabidopsis or from passion fruit?

Yuan-Yeu Yau 1. Had the *reference sequence* been used to do any experiment in a publication and worked? (reference of the paper?)

such as: Plant Cell. 2005 May; 17(5): 1467–1481. doi: 10.1105/tpc.104.030155

2. According to my knowledge, you are using passion fruits in your research. So, did this 'total RNA' you isolated to make your cDNA is from Arabidopsis or from passion fruit?

From Passion fruit

Hafiz Muhammad Rizwan < From passion fruit...>

Then, of course, you might get some different bases or amino acids from the Arabidopsis one. They are from different plant species. Unless the gene is very conserve (i.e. 100%), then, the genes will match perfectly. Just make sure your passion fruit copy is perfectly sequenced before you depot it into NCBI or use them.

Except reported from Arabidopsis, is any other plant species (*X*) also reported this gene sequence? If so, you can do an alignment of the gene sequences between Arabidopsis and plant species X, to see how many mismatches between them. You might need this info for your future paper too.

By the way, have you done the root GUS staining yet? (from another thread discussion). Got a result??

Yuan-Yeu Yau Then, of course, you might get some different bases or amino acids from the Arabidopsis one. They are from different plant species. Unless the gene is very conserve (i.e. 100%), then, the genes will match perfectly. Just make sure your passion fruit copy is perfectly sequenced before you use them.

as i am working on two varieties Yellow and purple. Before I successfully completed yellow cloning, I am now working on purple (but will do transformation together for both yellow and purple, as it will be convenient), even the Purple RNA quality >cDNA >PCR mix >PCR conditions are the same, but it's not 100% cloning results, and then yesterday i did cDNA amplification and sent the PCR amplified product to the company because i was want to make sure where is the problem; whether in the amplification or in TA cloning, so today i got the sequence results but when i did alignments then the amplification results are not good (attached file), so mean that my amplification is not 100%, so i will try again by using fresh RNA>cDNA then next,

But it made me curious, Do these two cDNA varieties need the same or different amplification conditions for the same target gene?If it is different, then why is it so?

By the way, have you done the root GUS staining yet? (from another thread discussion). Got a result??

I have not done rooting GUS staining because I am busy with other experiments, and I am considering to perform GFP>qPCR by using a negative control plant and a transformed sample showing a band, so whether they have GFP expression in the transformed plant compared to the negative control plant, then it should be used for GUS staining

Hafiz Muhammad Rizwan

(1) What did you mean by "but it's not 100% cloning results"?

(2) Except reported from Arabidopsis, is any other plant species (*X*) also reported this gene sequence? If so, you can do an alignment of the gene sequences between Arabidopsis and plant species X, to see how many mismatches between them. You might need this info for your future paper anyway.

(3) < But it made me curious, do these two cDNA varieties need the same or different amplification conditions for the same target gene? If it is different, then why is it so? >

If they have a similar length of cDNA, and only a few base changes, then I don't think you need to adjust PCR amplification condition. If there are heavily changes in an area, which causes -GC- rich, then you might need to adjust your PCR Tm value to be higher.

Hafiz Muhammad Rizwan

Look like you are sequencing a PCR product (fragment). Originally, I thought that you cloned your PCR product (fragment) into to a TA-vector first, then, into E. coli, then select a few clones from plate to do sequencing. But I think the PCR product should be homogenous too for sequencing.

Yuan-Yeu Yau (1) What did you mean by "but it's not 100% cloning results"?

PCR fragment into to a TA-vector first, then, into e. coli, then selected a few clones from it to be sequenced and that after alignment they have no 100 %identity in alignments, so i called then no 100% cloning results,

(2) Except reported from Arabidopsis, is any other plant species (*X*) also reported this gene sequence? If so, you can do an alignment of the gene sequences between Arabidopsis and plant species X, to see how many mismatches between them. You might need this info for your future paper anyway.

For that, i will have to search and need more time, before the reference sequences which i am using are based on Arabidopsis only.

If they have a similar length of cDNA, and only a few base changes, then I don't think you need to adjust PCR amplification condition. If there are heavily changes in an area, which causes -GC- rich, then you might need to adjust your PCR Tm value to be higher.

They have similar length cDNA (933 bp), but have heavily changed in an area, so will try to adjust by running gradient and by optimizing PCR conditions, also will try to use fresh cDNA, ( as before using 6 months old cDNA stored at -80, so maybe its effects)

Yuan-Yeu Yau Look like you are sequencing a PCR product (fragment)

t I only did PCR product (fragment) sequencing yesterday to make sure whether the PCR amplification)

before that, I always did sequencing after I cloned PCR fragment into to a TA-vector first, then, into e. coli, then select a few clones from it to be sequenced.

Hafiz Muhammad Rizwan < PCR fragment into to a TA-vector first, then, into e. coli, then selected a few clones from it to be sequenced and that after alignment they have no 100 %identity in alignments, so i called then no 100% cloning results,..>

Do you know this gene is present in the genome as a single gene, or it has a family, gene family, and with some degree of DNA sequence difference between them? If a gene family, you might amplified different members of the gene family and got a non-homogeneous PCR product.

Hafiz Muhammad Rizwan < They have similar length cDNA (933 bp), but have heavily changed in an area...>

I am surprised to hear that ("heavily changed of a DNA sequence in an area" ) between 2 varieties.

Kindly check https://www.promegaconnections.com/a-quick-method-for-a-tailing-pcr-products/

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

And http://tools.thermofisher.com/content/sfs/manuals/topota_man.pdf

Chinaza Godswill Awuchi Thanks for sharing,

Hafiz Muhammad Rizwan

So, do you figure out " if this gene is present in the genome as a single gene, or it has a family, gene family (with some degree of DNA sequence difference between them)? Or, even pseudogenes? So, how is the new sequencing results look like?

Yuan-Yeu Yau So, do you figure out " if this gene is present in the genome as a single gene, or it has a family, gene family (with some degree of DNA sequence difference between them)? Or, even pseudogenes? So, how is the new sequencing results look like?

Sir, This gene belongs to CER.

The new results show mismatches in amino acids alignment with the reference gene, maybe the amplification was not successful,,

So I will try again and will update you with the results later.