Dear Muhammad

I can try to help you if you explain to me the different steps you are following.

Which polymerase are you using to amplify your gene? What is the size of your gene? What's the percentage of GC of your amplicon?

When you say that the vector is not ligating, do you mean that your gene is not ligated in your vector or does it mean that your ligation protocol is not working?

I Can help you as well if you explain the steps you followed. I would like to know the Primer length. GC% and Tm that you have used. Which RE site you have in your gene of interest.

I suspect there are some technical problems here and not an issue with it being an impossible gene to clone. There would be no obvious reason the gene can not be amplified unless it had extraordinarily unusual DNA sequence such as very strong secondary structure (and even that would be surprising). As the others have said, provide more detail and perhaps you can get some useful suggestions, also be sure to include what controls you have done and what the results were.

ok, let me gather all the attempts i have made during last year. The story is really boring and disappointing. I will try my best to put in all the info:

The Rose transcriptome gene ARHL44646 with an astonishing length of 3141bp. My Prof. told me to clone only the CDS region that is 2340bp.

I started with designing primer pair with restriction site. PCR results with Taq-mix were not so good so i had to cut the band and purify. The conc. was so low. After 5-6 attempts, i managed to get 58ng/ul conc.. I started ligating it to pSuper 1300+ vector. It failed with no colonies. Tried 3 times. Then changed the primers with higher Tm, and followed the same steps, results were the same.

Then I shifted to T-vectors. I tried about 7 different primers at different sites out of the CDS. The Taq-mix was really bad, so I changed to Phanta (super fidelity) Mix. Amplication was a bit better, and conc. too was good. However, result was the same with pMD-18t vector. I tried other vectors like pCloneEz, Blunt kit, and pGem-t vectors. None of them gave me any hope.

At the end, sometimes I have no colonies at all, sometimes colonies with wrong gene, sometimes colonies with empty vector. I usually try atleast picking 48 colonies, and go upto 96. Everytime no hope. Just imagine me trying for about 1 year.. I also tried the cDNA from different methods, and different tissues, different stages. No hope.

Hope you guys got an idea.. If you need more info, plz let me know. Thanks alot

Which enzyme are you using ? Are you sure they are not present in your cDNA ? How far the restriction sites are from each other ?

For the pSuper1300+ vector (also known as pCAMBIA1300+), i used Xba1 and Spe1. Uploading the photo

The problem you have is that XbaI and SpeI generate compatible ends meaning that your plasmid can self-ligate. I advise you to change the enzymes e.g. HindIII/BamHI work well.

Muhammad Owais Shahid - a few missing details from your description. What is your template for amplification, are you amplifying from genomic DNA, or doing RT-PCR from an mRNA pool, or from an existing clone, or a cDNA library?

It seems to me that that main problem is the PCR step. After PCR you should never be limiting for DNA with which to clone, something is not going well at that step. Have you tried taking a small pool of your amplified product as template and to reamplify it so that you have plenty of DNA to work with?

When trying with the T-vectors, were you tailing your product or just ligating directly? You do realize that high fidelity polymerases don't leave T-tails?

# I was amplifying from cDNA, not gDNA ( BTW i have used gDNA in cloning the pre-miRNA, which was a piece of cake)

#Yes, I have tried that too. I amplified first, then re-amplified the resulted product, and thus I had a very bright band and good concentration, but didn't helped. My professor told me not to do that again as it will create unwanted nucleotides in the fragment, and thus you will not get the appropriate clone

# I used to ligate the t-vectors as mentioned in their protocols (after PCR product purification, i directly followed the ligation protocol). I don't know what tailing means.

Regarding the reamplification, so long as you are using high fidelity polymerase then it will be ok. Your professor is correct that reamplifying with Taq is liable to lead to mutations, but not so much with high fidelity polymerase. I would certainly try that again with Phanta.

If your primers are designed with a restriction site near the terminus (hopefully you have a left a few bases extra at each end for spacer), then digestion and ligation of your PCR product is usually the best. But you MUST have ample DNA to do this. I would begin with at least 100ng of amplified product. Most people gel purify after digestion, but I usually tried to avoid that because you can get UV damage if you aren't careful and frequently there is notable loss in DNA quantity after gel purification. But you should use whatever methods you are most comfortable with.

Regarding t-vectors and and tailing, Taq polymerase generates DNA fragments with a single A overhang at each 5' end, so a T-vector would be prepared with a protruding T residue (hence T-vector) so that the the insert and vector can ligate with increased efficiency. However High fidelity polymerases do NOT generate A overhangs with PCR so their product can not be used directly in TA cloning. But tailing is a procedure to add the terminal residue to PCR product prior to ligation.

great Sir, that was a nice information

TA cloning may fail because A is not always added with good efficiency. It depends on the last 3 nucleotides of the amplified DNA fragment. (see: Modulation of Non-Templated Nucleotide Addition by TaqDNA Polymerase:

Primer Modifications that Facilitate Genotyping

BioTechniques 20:1004-1010 (June 1996)). Try cloning into one of TOPO-vectors from Invitrogen first, and then re-clone insert into your vector

I successfully cloned my target gene to the T-vector, and the protein translation looks quiet good. Now problem after problem, mess after mess, I have to re-ligate this plasmid to another vector (pCambia 1300+) which is not working now.

The plasmid i extracted from the T-vector has many bands in the amplification PCR, don't know the problem is with primers or what.... One year wasted in this mess

As I said, the gene is cloned to T-vector successfully, but it is still not ligating to the florescence GFP vector (pCambia 1300+)... Its a big discouragement. I have been trying to do this since last year... I think i will be thrown out of the lab forever.

if you want some suggestions of things to try, please give us more details. When you say it is not ligating, we don't know if there is a problem with your source DNA, your ligations, your transformations etc. The more details you provide about the results and the controls you have done, the easier it is too diagnose.

Why do you run PCR on the T-vector with your insert? You do not have restriction sites to re-clone insert using endonucleases? More than one band in PCR with a plasmid as a template means too much of the template, or REALLY REALLY bad primers.

Hello dear I have some good ideas about cloning, It may help (In shaa Allah) if you didn't get your correct colonies till now.

At first, this is really long gen which is not good at all to use Taq for amplifying, specially its not high fidelity polymerase, I really recommend you to use high fidelity one.

Second, please don't use compatible Enzymes for double digestion (like Xbai/Spei or Bglii/BamHi), this will result in self ligation and wrong Alignment for your insertion.

Third, please use high quality vector and insertion by making PCR for example in 6 tubes (50 uL each) and run gell and get your band, extract it, then go to ethanol ppt. same as vector after miniprep good amount, go to ethanol ppt .

4th, add de-phosphorylation enzyme with digestion of vector or after and purify again using ethanol. (be attention about buffer condition, star activity and so on).

5th, use good (Fresh) ligase or Ligation mix and use it for overnight for example.

6th, transform as usual, however if you used to get few colonies you may go to ethanol ppt again after Ligation and use the purified ligation for your transformation.

7th, you can check if you have correct ligation by using colony PCR primers with Ligation product as a temple.

8th, you may need to insert your gene into ineducable promoter to clone it without any expression (if it toxic to your cells). , or to divide it into two parts and insert one by one.

Good Luck :)

Thank you brother Mohamed Shehata , I will your suggestions into account. I have tried certain things:

1) For this long transcript, i have used various polymerases and mixes, the high fidelity polymerase was the best. It helped me to ligate the gene to T-vector. Also, I have used A-tailing with taq polyemerase, that gave me a clone too

2) For the compatibility, I followed the principle that 'select 2 restriction enzyme sites in the vector which are not there in the gene'.. Then i searched for the 'list of compatible restriction enzymes'. So i selected XbaI-SpeI first, and then SmaI-SalI combination. For both, there are no results. Now you said 'don't use compatible Enzymes for double digestion', this statement is new for me, could you please elaborate it....

3) As far as ethanol ppt in DNA recovery is concerned, i have tried that too.. And, i also tried DNA recovery (DNA purification) without ethanol.. (because it is believed to be used with genes less than 1kb)

4) I will take this point into account for sure

5) I have tried ligation mix for overnight.. However, fresh ligate is not possible in our poor lab

6) I will take this point into account for sure

7) I always do the colony confirmation PCR by using either 2 vector primers or 1 gene and 1 vector primer. However the gene and vector primers are not compatible due to much difference in Tm

8) If i am not wrong, I think by ineducable promoter you mean the T-vector types.. I have already succeeded that. Otherwise i need to search what are these vectors

Thank you brother. May God Bless you...