9 replies to this topic

[Kami23]

Hi all,

I was  just wondering if anyone can help me.  I have been amplifying a gene in three fragments and so far two out of the three have been successful.  The problem is the C-Terminal end which just refuses to work. Apparently the primers are fine (as I came into the project after the primers were designed) but on my gel I have noticed a bright band which is tiny in size.  Do you think this could be primer dimer or non-specific amplification? Im trying to see if my supervisor will let me design my own primers but they seem convinced that that would do no good.  Its not just me, a college of mine cant get it to work either...

Any suggestions would be greatly appreciated!

Bex

P.s the two fragments that have worked were 2 and 3 kb but the one that wont work is 4kb... I was also wondering if the size could be a problem?

Edited by Kami23, 27 January 2009 - 07:49 AM.

[swanny]

Do you have a gel image for us?

[T C]

Hello

1. I would suggest that you chk the oligos first, if they are wromgly engineered, you are in a better situation to demand for fresh oligos.

2. Try gradient PCR and see if the Tm taht you are workign at is fine.  As a thumb rule, 4 degrees less thn the lower Tm works.  make sure the Tm of the oligos is close to each other.

3.  Whats yr template, if its a BAC clone, you can make a library and pick up a big chunk from there.

Hope it helps

TC

[BioBus]

also try nested PCR .
if you have BAC , r you also intetrested in try other way?

sth like : RT or galk

http://www.biomedcen...1471-213X/8/119

[swanny]

Have a look at the sequence of the C-terminal portion. If the GC content is high, try adding betaine (10% of reaction volume) or DMSO (1-5%) and see what happens. Alternately, you could try titrating the Mg2+ and see if you get a product, but be aware that increased Mg can lead to errors in the sequence.

Just how big is this mysterious band?

[Kami23]

swanny, on Jan 29 2009, 07:06 AM, said:

Have a look at the sequence of the C-terminal portion. If the GC content is high, try adding betaine (10% of reaction volume) or DMSO (1-5%) and see what happens. Alternately, you could try titrating the Mg2+ and see if you get a product, but be aware that increased Mg can lead to errors in the sequence.

Just how big is this mysterious band?

Sorry its taken so long to reply.  It a 4kb fragment amplified from human cDNA which i know is fresh because I did a control.  I have tried the DMSO thing and all possible concentrations of Mg2+.  I am using the advantage2 long pcr kit as changes would mean we have to discard the product.  Im at a complete loss! I know it is most likely the primers but convincing people will be the tricky part.

[pcrboy]

you dont need DMSO if its not GC rich, and i suspect that isnt the case. next, 4kb is quite a large band, i would suspect you would have trouble, but not a big one. I agree with the earlier advice by trying to play around with the temperature. if u think its the primer issues double check these parameters: both are close in Tm, primers are specific in their design to the target (simple blast), and end in G/C (for enhanced binding).  i am more concerned about the tiny bright band, primer dimer will usually not give you a bright band. i suspect its non-specific binding (the Blast will tell you how likely it will hit another transcript), and to fix that, you will need to raise annealing temperature.

[swanny]

Have you tried a touchdown PCR?

[AquaPlasmid]

What's your extension time? I once ran a primer pair for 3 amplicons (about 2000, 1000, and 400 bp), and at 30s I got some 1000 and strong 400 but not 2000, at 60s all three bands showed up with the 2000 the brightest. Most polymerases are said to read 1000 bp per minute.

[Kami23]

AquaPlasmid, on Jan 30 2009, 02:31 AM, said:

What's your extension time? I once ran a primer pair for 3 amplicons (about 2000, 1000, and 400 bp), and at 30s I got some 1000 and strong 400 but not 2000, at 60s all three bands showed up with the 2000 the brightest. Most polymerases are said to read 1000 bp per minute.

Yep Swanny I did but it didnt give me anything... and my extension time per cycle is 5 mins with a final extension of 7 mins...