PLease Please I need help with my PCR! Greatly appreciated!

[yasamino]

Hello Everyone,
I am new to this website and I hope I would be satisfied with the help I get for my first question on this forum Any help with this would be great since I am really overly stressed with this now!
Ok here it goes,
I did a PCR for my UB2 gene (if sequence required let me know and i can post it as well) and the following were my primers along with the rest of the PCR details that I followed.

Primers (in brackets are restrictions sites)

Forward Primer (NdeI)
5’ (c a t a t g) a t g g t g g a c t t g g c t a g g g t 3’
Reverse Primer (XhoI)
5’ (c t c g a g) t t a g c t g g a c a a c a g c t t t t c a 3’
Forward: %GC: 55, Size: 20 bases, Tm: 60.77C
Reverse: %GC: 40.91, Size: 22bases, Tm: 60.05C

PCR Components

PCR kit is from Bioshop
10X buffer-2ul
25mM MgCl2-1.6ul
10mM dNTP-0.75ul
10uM Forward Primer-0.5ul
10uM Reverse Primer-0.5ul
Taq-0.3ul
cDNA template-4ul (400ng/uL is template concentration)
dH2O-add up to 20ul

PCR Conditions

94C-4min
94C-45sec
Anneal (I did a gradient of 52-62C)-30sec
72C-30sec go to 2 repeat 30 times
72C-5min
4-for ever

My product size is suppose to be 585bp. I have attached a gel image of the PCR

08.24.2011 UB2 PCR w changes 08.23.2011.jpg

(20.97 KiB) Not downloaded yet

The lanes of the gel image correspond to the following
From Left
lane 1 - 1kb ladder
lane 2- skipped
lane 3- 53.7
lane 4- skipped
lane 5- 54.8
lane 6- skipped
lane 7- 56.3
lane 8- skipped
lane 9- 58.0
lane 10- skipped
lane 11- 59.4
lane 12- skipped
lane 13- 60.5
As can be seen in the gel image, my results are very faint bands and are unspecific (two bands in lanes 5 and lane 7 I think). The second band at the bottom I would think is the right size (585bp) since it is right above the bright 500bp band in the 1kb ladder. Am I right? So please suggestions as to how I can manipulate my PCR to get better more clear brighter bands? Am I on the right track and what am I doing wrong? All suggestions are welcome so please do suggest your thoughts

Thanks In advance to all of you!

[JackBean]

Shouldn't you have some overhang on the primers, so that the RE will be able to cut your product?

As of the PCR, I would just cut the lower band and not bother with the rest.
What is your template?

[greatmicrobiologist]

I want to know the RE you have used in PCR. The patterns are correct as it seems. I think there is some problem in RE along with Denaturation and Reannealing. Procedure is correct as it seems. In your final conclusion, you have mentioned about the 585bp of lane 5 or 7? I am little uncleared about it you have mentioned.

thanks.

[JackBean]

Restriction endonuclease used in PCR?

[greatmicrobiologist]

ooops i am sorry. I was thinking of different. My fault.

[antonyviridae]

From what I can see, your reaction looks good. Some suggestions that I have are:
-Don't use a gradient for the annealing. One temperature two-four degrees lower than your lowest primer Tm will suffice. Doing this might also get rid of those non specific bands.
-You need at least two-four nucleotides in front of RE sites because the enzymes need a 5` region to sit on to cut properly.
-Sometimes the final volume of a PCR reaction is important. You could try using a higher volume, maybe 25 or 50uL.
-Lastly, increasing your primer concentration might also help.

[JackBean]

increasing the volume of reaction? I thought, that it's better to have smaller volume, since the temperature changes are faster and more accurate.

[antonyviridae]

increasing the volume of reaction? I thought, that it's better to have smaller volume, since the temperature changes are faster and more accurate.

Though it may be true that a smaller volume would help with temperature changes, increasing the reaction volume also increases the amount of reagents that you have in the reaction. They are still at the same diluted factor but it gives your pcr a better chance at working. I know for my baculovirus work, that when I was using a volume of 20uL nothing would work but when I increased it to 50uL I was getting very good amplification. Also with the temperature thing, if you use an older style thermocycler the temp. changes are more gradual which would pretty much heat and cool your reaction accuratly no matter what volume it is.

[merv]

There's loads of things you can do to optimise PCR.

From my recollection, in order of preference, if you still need help on this:

0. Do a control reaction to test all the variables (taq, dNTP's especially, to ensure the PCR worked) - (usually someone can offer you actin primers and template at known molarity).
1. Mg concentration is key - alter this, use the temperature that gave you the best result.
2. You are using cDNA - does this mean a crude RT prep? You could do a DNA clean up to remove any proteins (a 50:50 Phenol/chloroform say 100-500ul , vortex, spin 10mins, pipette of the aqueous phase to a fresh tube. dispose of phenol carefully according to local rules (evap in a chemicals fumehood is best). Ethanol precipitate the DNA using standard protocols in Mol Biol - as your target is small, use some carrier DNA to ensure you dont lose the precipate - red-pellet precipation carriers are on the market (try SigmaAldrich).
3. Use less DNA- you will probably get better results, and save your precuious DNA.
4. Use thin wall tubes for PCR - and cut your cycle hold times (means you can get faster sample to result times - great if you have insomnia anyway/ you can shorter breaks).
5. eliminate the final step of 72oC - it is rarely needed, and if you are cloning it can cause damage to the phosphate ends and your cDNA cloning might not work.
6. reduce the initial heating step time as this can damage you enzyme.
7. Avoid too much enzyme- always use a master mix of enzyme which is well vortexed to mix- otherwise glycerol in the reaction can be variable across your samples and too high. Always keep your reactions on ice. Sometimes batches of enzyme vary and have variable shelf lives...
8. Try hot-start PCR if thats not working well.
9. Consider the possibility that your larger bands are alternative spliced variants and clone and sequence them too. But beware that PCR often creates bizarre unnatural hybrid molecules which are entirely artefact.

If non of this helps, either your technique is wrong, or cut your losses and redesign your primers. I think that covers it!