Hello there..!!

I have gone through your question..and found that..the parameters what you are employing can be optimize....

1. Take more amount approx..20-30 ng

2. Increase primer concentration upto 10 micromolar

3. and check your extension time for PCR (1 min/kB)*...* important

Hope it will work for you..............//

Are you talking about eukaryotic gene ? 

Because u indicated cDNA (which is RNA to DNA conversion) in which you may used random primer, which will not produce complete 4000 bp length.

if you do pcr with cDNA then its tough to get a full length of your Gene.

You can use primer specific for your gene target with Special PCR mix, like Long Range PCR kit in Qiagen, where special enzyme for longer length of product upto 10kb can be obtained.

If i misunderstood your query means, kindly give me a clear query? to my mail

[email protected]

if my explanation suits you, thanks.

For cDNA, you need to optimize 2 steps of your study, RT and PCR.

In the RT, you need to use a enzyme that might process long fragment and increase the time of elongation during RT, in order to process the 4Kb. I use superscript III. Becareful, with this enzyme do not work in classical temperature...

For PCR, use a long range taq polymerase. As say, use a ratio of 1kb / min or below in the elongation step.  Sometime, the long range enzyme has an optimal elongation temperature around 68°C and not 72°C. Use it. Becareful about the amount of dNTP. they are very important.

thank you for the information. I had already increase the concentration of primer until 1mikromolar, but I just only get a smear band again and the size is not in the target size. for elongation step I had set 1kb/min. I was a little bit confused, is that problem in my cDNA? Is it probably the cDNA does not produce complete length?

1. Is 4 kb: genomic DNA (exons+ introns) or cDNA (exons)? If the 4-kb is the length of genomic DNA, the size of the PCR product can be smaller when cDNA is used as PCR  template

2. If you are working on the cDNA, make sure your gene is expressed (mRNA) during the particular development stage you used to isolated the total RNA. cDNA is derived from mRNA. Some genes only express at certain development stages.

3. According to its booklet (see attachment), KOD FX enables the following amplifications (Maximum): 40kb from lambda phage DNA, 24kb from human genomic DNA, and 13.5kb from cDNA. So, to amplify a 4-kb product should be no problem.

4. From the booklet, it suggests to use 0.3 uM primer (FINAL concentration); you were using 0.1 uM (and was this the 'stock' concentration or 'final' concentration)?

5. What are the Tm of your primers?

6. Try to increase cDNA amount as suggested by Ramnath

Thank you Yuan-Yeu Yau.

1. 4 kb : cDNA (exon), 2. yes, 3. yes, 4. I'm sorry, I wrong write the volume my stock, so the final concentration each primer is 0.3uM (same in protocol KOD), 5. ™ : 67C(F), 68C (R). 6. Ok.

thank you

Thank you for your answers. By the way:

1. What kind of primers are you using to amplify cDNA template? Gene-specific primers? Make sure your primers were designed based on the 'cording (exon)' regions, not the 'intron' regions to amplify cDNA template.

2. 2-4% DMSO can be added to PCR reaction to increase the accuracy and efficiency of PCR amplification, in case your gene is GC-rich.

3. Include positive controls for PCR.

Also, the 4-kb cDNA is pretty long, and Stephane raised a good point, you should check whether your RT-PCR kit has the ability to synthesize full-length 4-kb cDNA under their protocol.

What kind of RT-PCR kit are you using?

Try using the protocol in the attached publication

In addition to the comments above: You should check the RNA quality. To reverse transcribe 4kb full length transcripts you need RNA of high quality- maybe rather from cell lines than from tissues etc

Following up on Yuan-Yeu Yau I would also recommed to add a PCR additive.

I agree that your first choice should be DMSO  which is thought to reduce secondary structure and is particularly useful for GC rich templates.

Another known PCR additive is BSA (Bovine Serum Albumin). BSA increases PCR yields from low purity templates. It also prevents adhesion of enzymes to the reaction tubes and tip surfaces.

I worked with both PCR additives in different type of experiments and I received very good results. 

First of all your cDNA quality must be ok. To amplify whole sequence, Long -Range PCR is the best option. You can use cammercial kit but it cost too much for you, instead you can design your LR-PCR.  Methodology is availbale in this article (attached):

Novel Complex Re-Arrangement of ARG1 commonly shared by unrelated patients with Hyperargininemia.

good luck.

Thank you for all the advice.

I try the PCR again with 3%DMSO and increase the conc of cDNA, but  I can not get the band (no smear too). I'm using gene specific primer (only exon)

Im using ReverTra Ace® qPCR RT Kit.

how to check the RNA quality?

thanks

Take a look of this: Barnes W. M. 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from l bacteriophage templates. Proc. Natl. Acad. Sci. 91:2216-2220. Maybe it would be helpful.

In the protocol of ReverTra Toyobo you attached, at RT step of '37C incubation', the protocol suggests using 15 minutes. At this step the protocol (in its Note) also suggested that 'This 37C incubation step can be prolonged up to 1 hour.'. Since your cDNA is pretty long, you should use a prolonged incubation time. How long of the reaction time did you use?

Dear Yuan-Yeu Yau

I'm sorry I wrong attached the protocol of RT. 

this one in correct : http://www.toyobo-global.com/seihin/xr/lifescience/products/mod_001.html

Firstly, I was isolation RNA from the cell line using Trizol Reagent. After that RT using revertra toyobo. 30ºC, 10 min, 42ºC, 20-60 min, 99ºC, 5 min

thanks

dear Kenzy Peña,

I have already read the paper, that paper showed about Pfu and Taq polymerase and that paper also recommend to combination two enzyme.. But I think in this case I had already use KOD that more stable in high temperature and less mutation than Pfu and Taq polymerase. http://www.toyobo-global.com/seihin/xr/lifescience/technology/001.html.

anyway thank ^^

Dear Dyaningtyas,

I agree with you, your enzyme is better than Pfu and Taq but maybe the addition of two enzymes (one with 5-3' activity and the other with 3-5' activity) is the answer. Maybe you can use your KDO enzyme and other to try to improve your essay. In the Protocol 13 (called Long PCR) from the Molecular Cloning book (by Sambrook J. and Russell D.W., volume 2) the usage of two enzymes to amplify fragments up to 25 kb is suggested. Also, they list some important considerations to amplify big fragments like: quality of DNA, primers size and annealing temperature.

Regards.

dear Kenzy Peña,

i'll try it. thanks

Hi Dyaningtyas,

Thanks for the re-attached protocol. Just curious, what kind of primer (oligo(dT) or gene-specific primer) did you use for RT reaction?

Also, I read the protocol, in the Example 3, for 42oC, 30 min, they can generate at least 14-kb cDNA (and then PCR product); so for 4-kb cDNA like yours should be no problem using this kit if things go as it supposes to go. I am wondering whether or not you have another set of primers (near the 3' end) which can amplify a short part of the cDNA. So we will know if the target cDNA is generated at all, no matter a full-length or a partial one.

Dear All I have to make a PCR with my designed primers bringing the restriction enzyme site at the 3' and 5'. However I am having problem as well for getting cDNA band for my 4000bp cDNA. I have tried to make several PCR with 12 different Tannealing but I could't get any band. I have tried to use for my cDNA two primers of control, beta-actin, and they worked. Which is the problem, my primers? When you calculate the Ta of your primers you consider just the overlapping sequence or the entire sequence with the restriction site sequence included? I am using the Q5 high fidelity DNA polymarase that is suitable for long cDNA, whereas I used for my RT random primers and the M-MLV reverse transcriptase able to cover more than 4kb length. does anyone know which may be the problem?

I have tried different conditions and kits but i could not get any band. Finally i could amplified 4000 bp from cell cDNA by extension of the primers length. The primers should be design for your gene of interest with extension of 20 bp that are complementary to the ends of the linearized vector. I also recommend Takara kit for PCR and cloning of long DNA fragments.

(https://www.takarabio.com/learning-centers/cloning?gclid=Cj0KCQjwjbveBRDVARIsAKxH7vkhUsWNbE8z6xYsenr8YDWgh0eRKpZjqJi4evVxtPgxD4Eg6XtKFe0aAqDxEALw_wcB)