Problems (unspecific products) with PCR to determine the size of cDNA inserts - (Jun/21/2006 )

Hi there,

I constructed a cDNA library using the CloneMiner cDNA Library Construction Kit (Invitrogen, Cat. No. 18249-029) (see attachment CloneMiner method). I am currently determining the size of my cDNA inserts by means of PCR with the following sequencing primers (see attachment Recombination Region), which are recommended by the CloneMiner kit to sequence the inserts:

Forward primer (proximal to attL1):

M13Forward(-20): 5'-GTA AAA CGA CGG CCA G-3'

Reverse primer (proximal to attL2):

M13Reverse: 5'-CAG GAA ACA GCT ATG AC-3'


Nevertheless, I have had some problems with the PCR since I have gotten some unspecific products (see attachment PCR products). Because I obtain several PCR products I am not really sure which of them is the one derived from the cDNA insert nor the product derived from the vector (pDONR222). I am also attaching the pDONR222 map.

The PCR mix conditions per reaction (final concentration) I have been using during this procedure is the following:

Buffer 10X (0.8X)
MgCl2 (1.5 mM)
dNTP mix (0.15 mM)
Primer forward (0.10 uM)
Primer reverse (0.10uM)
Bovine serum albumine-BSA- (0.8 ug/ul)
Taq polymerase (0.5 units)

The PCR program conditions are as follow:

1) 94 C for 1 min
2) 94 for 30 sec
3) 55 for 2 min
4) 75 for 2 min
5) go to step 2) 29 times
6) 75 for 5 min
7) 75 for 5 min



As you can see in the attachment PCR products, there are unspecific amplification as there are easily visible other bands other than the more intense one, which I think is the one that has the cDNA insert. But I am confused with this results because I do not understand nor interpret the visual pattern of the amplified pDONR222 (line D1) and the non-amplified pDONR222 (line D2). It is supposed that the entire vector alone has a size of 4718 bp. It is also supposed that the other lines have the cDNA insert, but I do not know how to discriminate it from the vector due to its unusual visual pattern.

What can I do further in order to eliminate the unspecific products and to find out the real PCR product of my entry clone so that I can determine the real size of the clone?

Thank you in advance for your help and suggestions.


Danilo

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Hi Danilo,

let me understand something: what did you use to start? I mean, did you use a mix of mRNA that you ligated to the polyA tail and then reverse transcribed? And is the extension temp suggested on the Taq datasheet?

Raffaela

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Hi Raffaela,

Thank you for your reply. In order to construct the cDNA library, I first isolated total RNA and then mRNA which was reverse transcribed into cDNA.

As to the PCR extension time, I did not take into account any suggestion of the Taq datasheet because the Taq que use is produce by ourselves.

Let me know if you need further information.

Danilo

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I read on page 43 that you can digest with Bsr GI. Why don't you try that? It should be easier and more reliable. The strange amplification of your plasmid could be due to several factors. Maybe you could try and increase the annealing and check if your DNA is clean enough.

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Hi again Raffaela,

I know I can use the BsrGI enzyme to determine the accurate size of my cDNA inserts, yet that cannot be appropiate for screening a lot of entry clones. I decided to perform PCR because is cheaper and I can evaluate hundred of clones. You know, PCR reagents are easier to find in a lab.
That is why I am using it, so my goal is to standardize the technique to screen a lot of clones at time.

I would really appreciate your recommendations regarding this matter.

Thak you,

Danilo

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In order to reduce the non specific bands and the smearing you see on your DNA, I would increase the annealing temperature (2 degrees) and decrease the amount of template (50ng is enough).
I would try with your vector first and when you have something decently clean, you can do it for the others as well

Good luck and let me know!

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Dear Raffaela,

I already modified the Tm of the primers whose initial Tm is 47 and 51 C. So, I increase it to 55 C. Regarding the amount of template, I used 2 ul of a clone diluted in 10 ul of sterile water.

Unfortunately I do not have enough vector to perform further probes. I will persist on this procedure anyway.

Danilo

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Well,

I cannot be of more help. Sorry!

Wish you the best with the following experiments!

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