How can I get the 4.5kb Human typeI procollagen alpha1 chain cDNA Via RT-PCR fr - Seeking Help (Nov/04/2005 )
Hello,everyone!
Now, I RT-PCR the cDNA of Human procollagen typeI Alpha1 chain. The Full length of this cDNA is 6.7kb, I just want to amplify the coding region of this cDNA(size:4.5kb). I have extracted the Total RNA from huamn skin, and electropheresis show The total RNA is intact! I, however, cannot get the fragments desired! the GC content of this fragment is 63%, and the Td predicted by Oligo6 is 110 centigrade degree!(may be too high for the Taq DNA Polymerase), I PCR it With glycerol with final concentration 5%, but ,nothing is detected via Argrose Gel electrophoresis, even unspecific bands! Now I am very Upset! Can you give me some recommendations!
Thanks! await your reply!
hi
for GC rich contents, i use eppendorf triple master or Phusion and that goes. but try to reduce tm of your primer...
[quote name='fred_33' date='Nov 4 2005, 08:04 PM' post='29959']
hi
for GC rich contents, i use eppendorf triple master or Phusion and that goes. but try to reduce tm of your primer...
[/quote
Thank you, Fred! Can you tell me which company provides the reagents you use,and how do you amplify your long fragments via RT-PCR. And how long is you desired fragment? What reaction procedure do you adopted in RT-PCR. Should I increase my melting temperature, for when I check my desired fragments via Oligo6, I find that the Tm of PCR product is 110 centigrade degree! so should I increase the melting temperature from 95 to 110 centigrade degree? I appreciate your suggestion!
It seems that no one is willing to help me!
I am very mad and discouraged for this problem now, for this project is very imperative.
Hi!
It would be more helpful if you provide more details concerning your PCR protocol, e.g. elongation time, GC content of your primers, have you tried a 'touch-down' PCR.... .
Cheers
Now, I RT-PCR the cDNA of Human procollagen typeI Alpha1 chain. The Full length of this cDNA is 6.7kb, I just want to amplify the coding region of this cDNA(size:4.5kb). I have extracted the Total RNA from huamn skin, and electropheresis show The total RNA is intact! I, however, cannot get the fragments desired! the GC content of this fragment is 63%, and the Td predicted by Oligo6 is 110 centigrade degree!(may be too high for the Taq DNA Polymerase), I PCR it With glycerol with final concentration 5%, but ,nothing is detected via Argrose Gel electrophoresis, even unspecific bands! Now I am very Upset! Can you give me some recommendations!
Thanks! await your reply!
110 centigrade degree is to high! I would try an annealing temperature between 50°C and 72°C. Try 55°C.
It would be more helpful if you provide more details concerning your PCR protocol, e.g. elongation time, GC content of your primers, have you tried a 'touch-down' PCR.... .
Cheers
Hi Bomber,
I Have adopted the TD PCR, and my Melting Temprature is 95℃ I denature the tepmlate and primer at 95℃ for 2min first, then 95℃ 15second, aneal the template and primer at 66~56℃ with each cycle decreasing 1℃ for 30sec , extend DNA at 68℃ (for the desired fragment is 4.5kb Length) for 5min, 10cycles, then proceed the system in next 20cycles with the program: denaturation at 95℃ for 15 sec, anealing at 56℃ for 30sec , elongation at 68℃ for 5min, then at 68 for extensive elongation for 15min, PCR product is stored at 4℃.
Oligo6 show that the Tm of my PCR product is110centigrade degree, should I improve my melting tempature from 95 centigrade degree to 110 centigrade degree?
RT condition is: reverse transcriptease is :MMLV which has RNaseH activity and at 37 has its full activity, MMLV is contained in the Kit provided by BBI. I proceed the RT reaction at 45 centigrade degree for 1.5Hour, because of the length(CDs:4.5kb, full lenth of the ProIα(1): 6.8Kb) and GC rich content(65%) of my fragment, I do it according to the guide of the protocol attached with the kit provided by the provider--BBI. I prime the mRNA with oligo(dT) and GSP(gene specific primer). I am sure my reagents has no problem!
should I continue to improve RT reaction Temprature? to what centigrade degree?
I do not adopt the control reaction, What mRNA affluent enough in Human skin could be used as a control ? how can I optimize my RT-PCR to get my product?
Hi!
Can you amplify anything out of your cDNA pool, something like GAPDH or something else?
Otherwise your RT-reaction might not work well. I would do a positive control for appropriate RT-reaction to exclude a malfunction of this reaction.
Another possibilty might be the another Polymerase (I suppose you use Taq....?). You could try to use a high accuracy polymerase for long fragments (Pfu is generally working nicely).
You could try this link.
http://www.stratagene.com/products/display...ct.aspx?pid=416
("Optimum temperature near 75°C
95% active after 1-hour incubation at 98°C").
Might be a possibility to work with an enzyme that can maybe handle very high melting temperatures.
Good luck!
Cheers
Can you amplify anything out of your cDNA pool, something like GAPDH or something else?
Otherwise your RT-reaction might not work well. I would do a positive control for appropriate RT-reaction to exclude a malfunction of this reaction.
Another possibilty might be the another Polymerase (I suppose you use Taq....?). You could try to use a high accuracy polymerase for long fragments (Pfu is generally working nicely).
You could try this link.
http://www.stratagene.com/products/display...ct.aspx?pid=416
("Optimum temperature near 75°C
95% active after 1-hour incubation at 98°C").
Might be a possibility to work with an enzyme that can maybe handle very high melting temperatures.
Good luck!
Cheers
Hi Bomber,
Thanks for your suggestion!
recently, I have tried the Superscript II reverse Transcription System provided by invitrogen, which could preserve full activity up to 50 centigrade degree, then I use the Pfx DNA polymerase to amplify my dsired DNA segment, but the PCR product displayed by Argrose gel electropheresis is smear, what a discouraged result! I nearly quit this expreiment!
I search several well-known biotechnique Company to find a good RT-PCR product which may solve my problem. But I find that hardly any product could meet my demand, as I have outlined above, the cDNA of my interest is 6.7kb long if I want to Reverse Transcribe the full length of it. Even If I USE The GSP as the RT primer, Its length, namly the length of CDs is 4.5kb, It is really a headache to me! and the GC content of this fragment is 65%,Some of the Reverse Transcriptease from Qiagen or Invitrogen or Roche, claimed that they are robust to Reverse transcribe a GC rich template, but when they RT the Gc rich template, they seems a liitle invalid to RT a long one. in a sipmle word is they can not RT a long (>4kb) GC rich template. It is really an unfortunately news to me! Do you know some products that could RT a long (>5kb) GC rich(GC content >65%). Or other effective methods to resolve this headache? Please tell me ! Thank you for your patient answer and your suggestions!
A colleague of mine succeeded in amplifying about 6 kb with Superscript III from Invitrogen @ 55°C, and her husband (who's working in another lab) succeeded in amplifying about 9 kb with Transcriptor from roche (don't know his conditions, but probably @ 50°C). Takara has an RT that they claim works @ 65°C, maybe you can try that one? (http://www.takarabioeurope.com/rt_pcr.html)
But the problem to me seems that you can not know if your RT has worked, because your PCR isn't optimised either.
If trying pfu, you better work with pfu turbo or pfu Ultra (also from stratagene). They are improved versions of the original. Also, for pfx from invitrogen, it works a lot better if you double the concentration of the PCR buffer...