problem with PCR --using fragment as template - (Apr/08/2005 )
Hi
I use a 520bp DNA fragment as template, with RE sites at the end. first i try directly to amplify it from gel-extraction products, but it appears a smear (very bright streak). sencond time i double digest it (Xhol 1 & EcoR1)and amplify with the same primers ,it turns out the same!
and the smear is larger than the template!
what's the reason and how should i do ?
thanks
ananda
the gel pic like this
hi
i get bad results from a gel extracted template. i did two things.
First i dilute my template to 20ng/µl and started with 10ng
second i increased by 5° the annealing temp.
If it's not sufficent you can add DMSO (to final 1% but i'm NOT sure of the concentration) and it reduces the non specific reactions.
hi,Fred
in fact the amount of my template is very low(though not quantitive), DMSO could help,i will have a try! thx
and i want to know is there anything related with the template itself?Could linear,short fragments be used as template
Go with fred's idea.
when ever you get a smear, that is not bad, that is way better than black and empty space !
raise your annealing temperature like he said, and if that is so high you get no product then try a few annealing temperatures in between.
If that first routine still gives a smear, then use the highest annealing temperature that gave you something, and set up reactions with extra additions of Mg++. (vary the Mg++ in 0.5 mM steps, most buffer mixes with Mg included already have 2-2.5 mM, and you often get an extra Mg++Cl stock).
http://ken.mitton.com
ok,,i will do it tomorrow
the problem is the Tm of my primer is 74 & 64 centigrade, it sounds too bad but i really amplify a specific band via RT-PCR, annealing temperature is 55!
the problem is the Tm of my primer is 74 & 64 centigrade, it sounds too bad but i really amplify a specific band via RT-PCR, annealing temperature is 55!
IF you have primers with extra overhangs, check the TM of just the portion that initially anneals to your template (not the overhang portion). Just be simple and use Tm est. = 4x(numberGC)+2x(numberAT)
Use the lowest Tm of the pair as a guide to test a first annealing temp. If Tm's are in the 55 range, I usually do not go below 55 to start. If Tm's are about 60-63, I start 3 degrees below that for anneal.
Most thermocyclers will also let you do touchdown PCR. program the first five cycles to start 5 degrees above a final anneal temp, and decrease the annealing temp, by 1 degree C per cycle, then complete PCR at a fixed temperature. Ie. start at 64, then 63, 62, 61, 60, 59 then have 25 cycles more of 59 anneal.
For any of the above methods: if your Tms are very different, do reactions with 1:1 (higher Tm primer : lower Tm primer) primer PLUS: 2:1 ratio (higher Tm primer: lower Tm primer) while keeping the same total primer amount.
good luck.
http://ken.mitton.com
hi, ananda,
i had the same problem two months ago---amplify my band from gel extraction with the same primers, but got only smears.
i tried everything but all failed. at last i found there's something wrong with the UV light used to cut the desired band. the UV is too potent and the DNA band was so seriously damaged that it could not be used as the template to amplify again with the same primers.
so my suggestion is : expose your gel under UV as short as possible. if there is only one band ( 500 bp) in your pcr product, don't use gel extraction!!! just use pcr purification kit to get 500 bp. to do this, you could run only a little fraction of your pcr reaction product on the gel to see if there is only one band. then use the clean 500 bp product to reamplify with the same primers.
good luck!
I use a 520bp DNA fragment as template, with RE sites at the end. first i try directly to amplify it from gel-extraction products, but it appears a smear (very bright streak). sencond time i double digest it (Xhol 1 & EcoR1)and amplify with the same primers ,it turns out the same!
and the smear is larger than the template!
what's the reason and how should i do ?
thanks
ananda
I'm interested in the last lines of your post, because it also happened to me: how it can be that the smear is larger than the template???
Cheers
W.
Use the lowest Tm of the pair as a guide to test a first annealing temp. If Tm's are in the 55 range, I usually do not go below 55 to start. If Tm's are about 60-63, I start 3 degrees below that for anneal.
Most thermocyclers will also let you do touchdown PCR. program the first five cycles to start 5 degrees above a final anneal temp, and decrease the annealing temp, by 1 degree C per cycle, then complete PCR at a fixed temperature. Ie. start at 64, then 63, 62, 61, 60, 59 then have 25 cycles more of 59 anneal.
For any of the above methods: if your Tms are very different, do reactions with 1:1 (higher Tm primer : lower Tm primer) primer PLUS: 2:1 ratio (higher Tm primer: lower Tm primer) while keeping the same total primer amount.
good luck.
http://ken.mitton.com
it's great! and i want to know is there any reason for the step down interval is 1 degree C, could 2 step such as first 64 then 59 work?
i don't understand the last line your post about the primer's amount. Could you do more explanation? and the total primer amount is so important that we have to keep it the same? sorry to trouble u.