5' race

[bushra12]

Dear all,
i m trying to do 5' race of plant genes... I'm using clontech kit..
in one case it worked and in one gene it is not working out.. i even synthesized new cDNA thinking my gene is expressed at low levels but still no bands!
what to do?

[relaxin]

Since the kit works for one gene, you should not have technical problem. Perhaps the one that does not work is already a full-length transcript. Alternatively, there may be degradation of mRNA. You may try again with a fresh prep of RNA.

[mchlbrmn]

With no details of what you did and what result you saw, you're less likely to get useful advice.
Can you PCR this gene from your cDNA template?
Do you get nothing, or multiple bands?
There could be secondary structure or a high GC stretch that is difficult to PCR through. I'm not familiar with your RACE protocol, but judging by other PCR a higher anneal temp (or lower if it's a primer problem), or a cosolvent (such as DMSO 3-10%, or betaine) may help.

[bushra12]

Since the kit works for one gene, you should not have technical problem. Perhaps the one that does not work is already a full-length transcript. Alternatively, there may be degradation of mRNA. You may try again with a fresh prep of RNA.

thanks for the reply, i have tried 3 different sources of RNA. i also lowered the Tm and was able to get multiple bands.. will try different Tms now.. btw can u suggest me hows Primescript RTase by takara if u have used it.. (as i used it for cDNA synthesis... its an MMLV variant as the one provided by clontech)..

[bushra12]

With no details of what you did and what result you saw, you're less likely to get useful advice.
Can you PCR this gene from your cDNA template?
Do you get nothing, or multiple bands?
There could be secondary structure or a high GC stretch that is difficult to PCR through. I'm not familiar with your RACE protocol, but judging by other PCR a higher anneal temp (or lower if it's a primer problem), or a cosolvent (such as DMSO 3-10%, or betaine) may help.

thanks for the response.. i cannot PCR amplify this gene as i have a partial EST.. since genomic resources for trees are scarce i dnt have much information... so I'm trying to complete this EST by 5' race. initially i got no bands at all but today i was able to get multiple bands after lowering the Tm... so ill try to change the Tm tomorrow as see what happens.. the GC content of the EST is fine (40-50%) so dnt expect that to be a problem...

[relaxin]

btw can u suggest me hows Primescript RTase by takara if u have used it.. (as i used it for cDNA synthesis... its an MMLV variant as the one provided by clontech)..

Clontech 5--RACE kit incude SMARTScribe™ Reverse Transcriptase. Why don't you use that? A variant may behave differently.

[bushra12]

btw can u suggest me hows Primescript RTase by takara if u have used it.. (as i used it for cDNA synthesis... its an MMLV variant as the one provided by clontech)..

Clontech 5--RACE kit incude SMARTScribe™ Reverse Transcriptase. Why don't you use that? A variant may behave differently.

that was not provided in the kit earlier... i can try that also but in the meantime.. i guess will need to optimize PCR conditions.. as now I'm getting multiple bands...

[mchlbrmn]

With no details of what you did and what result you saw, you're less likely to get useful advice.
Can you PCR this gene from your cDNA template?
Do you get nothing, or multiple bands?
There could be secondary structure or a high GC stretch that is difficult to PCR through. I'm not familiar with your RACE protocol, but judging by other PCR a higher anneal temp (or lower if it's a primer problem), or a cosolvent (such as DMSO 3-10%, or betaine) may help.

thanks for the response.. i cannot PCR amplify this gene as i have a partial EST.. since genomic resources for trees are scarce i dnt have much information... so I'm trying to complete this EST by 5' race. initially i got no bands at all but today i was able to get multiple bands after lowering the Tm... so ill try to change the Tm tomorrow as see what happens.. the GC content of the EST is fine (40-50%) so dnt expect that to be a problem...

I did not mean, "can you PCR the entire gene". I was asking if you could get a PCR product only based on the original EST, preferably with one of the RACE primers, as a control to show that you had enough template of that transcript to work with, and if a RACE primer was used that shows that one primer at least works under the given PCR conditions.
Multiple bands are not necessarily a bad result. Some genes have alternate start sites or alternate splices that would create more than one 5' length.
Even if the GC % of the EST is favorable, that doesn't give any indication of the GC% of other regions. I think 5' ends sometimes can have a high GC%.
I don't know what the best course is for you, and haven't looked at your protocol, but it can be possible to get data out of multiple bands even if some are nonspecific. Bands can be cut out of a gel, purified, cloned, and sequenced. In some cases it works to cut them out, PCR more (not too much, I think, or you may end up with multiple bands again) and sequence the clean PCR product if it is one clean band.

[bushra12]

mchlbrmn,
well actually i am strictly following the protocol provided in the manual.. i once got a neat band (expected size) at 65 degrees but the concentration was too low, so i decided to repeat that PCR under the same conditions.. but now i get a multiple bands all the time and the desired band is too faint to be extracted properly... this is the current situation now.. I'm trying to repeat the same PCR and not getting the same bands.. the bands of smaller size are more intense and have a very faint band at the expected size...

[mchlbrmn]

I'm not sure how you know what the correct size is, but here are some ideas to get around an extremely faint band.
The first thing that comes to mind for such a situation is to nest the PCR. Probably your procedure already does nested PCR, at least on one side, the known sequence side. You could try an additional nest step with a primer inside of the last 3' primer used. If the multiple bands are specific (alternate transcripts e.g.) this won't accomplish anything, but it would boost specificity and could get rid of nonspecific bands.

You try and could cut out the correct band and PCR more without purifying, if it is only a couple of nanograms and too little to gel purify. This might work best if some kind of low melt or 'biotechnology grade' agarose is used (designed to not inhibit enzymes), but can work with a small % of regular agarose in the PCR mix. Taq is relatively tolerant of impurities. I'm not sure how sensitive other proofreading enzymes are to agarose contamination. As I said before, you don't want to use too many cycles or it can become messy with miniscule traces of other bands being amplified also, or a smear coming up. Well, even if it is somewhat messy you will have boosted the amount of product and if you clone you would get pure clones and check for the correct one.

It's also possible that this unrepeatable, faint band is actually nonspecific also.

Even if you follow the protocol perfectly, if the starting template was weak or degraded the result may not be good. That is why I suggested conventional PCR on the EST sequence alone as a control to see if the template can be readily PCRed, or if it does contain the target message.

If you haven't already done so, you can call the kit manufacturer and, hopefully, speak to an expert in this specific protocol who may have advice.

[bushra12]

mchlbrmn
thanks for the response...
actually i compared the size of my est with sequences from other organisms and thus estimated the approx size... I'm thinking about extracting that band.. but not hoping much as it was a very faint band plus it was loaded on a regular 0.8% gel for checking pcr... i can't do nested per as i loaded all i had and only them i was able to see that band... since then i have been trying to get that band and repeating the same set of reaction and I'm getting either non specific bands or no bands at all...
i was able to amplify two genes completely from the same cDNA so I'm hoping that my cDNA is fine... the manufacturer dint help much as I'm using an older version of this kit.. which has now been replaced..

[mchlbrmn]

You can take the faint band, before or after purification, and PCR it a bit more to increase the concentration. Also, I would guess that the ends are more likely to vary from other species than the center of the transcript. Alternate splices are most prevalent near the termini, not to mention the untranslated region could vary more than the center. If you blotted the gel you cold probe to see which bands were correct, but that's too much work. I wonder if the bands could be checked by a quick PCR, if you have internal primers in the known area.
If there is a stop in the RT at a high GC region, a higher RT temp might help.
Just some ideas, you can weigh...