We want to construct a plasmid to over-express a protein in breast cancer cells, but the expression level of this protein and mRNA is low, and there are four variants of mRNA. Thus, we used nested PCR to amplificate the sequences of CDS region(924bp). However, when we used agarose gel electrophoresis of 1% concentration to test the products of PCR, the produts couldn't been separated, and we don't know why. And several students in our lab also encountered this problem.
Please provide more information: what is in the various lanes. With how much mRNA did you start the RT PCR procedure? Define "nested" in this case and describe the conditions.
how many cycles total are you running?
10 cycles of pcr is 1000 times more product so 2 amplifications each of 30 cycles is 10exp18 times more product so very soon the amount of product is huge and it is likely that amplimer to amplimer annealing rather than primer to amplimer is happening and the amplimer is just getting elongated. Doing nested pcr it is a good idea to dilute the first amplimer 1/100 to 1/1000 and then only run 10-20 cycles removing tubes every few cycles to get a feel for how many 2nd round cycles are needed for clean amplification
As my colleagues said above, you should provide more information about which lanes contain what products, what lengths do ladder bars represent, how many PCR iterations did you have, how many cycles did you use for every iteration, and how did you purify/dilute amplicons between iterations.
I tend to agree with Paul Rutland that you have amplimer to amplimer annealing here because the lower front of the smear is very clear, that means you have the smallest possible length for products. Is it by any chance the same as your expected amplicon length?
I can also think of a couple of different explanations for smears. The smears you have on this gel look like high-molecular genomic DNA for me. Probably you didn't dilute your DNA matrix enough before using it for PCR (did you use cDNA or genomic DNA to begin with?)
Also, did you check your primers with a positive control sample before you used them for the experiment? There is a possibility that one primer didn't anneal and you have one-sided amplification here, but usually in this case the smear runs lower.
We extracted total RNA and used 5ug to reverse(total volume 20uL), and got cDNA.
We designed primer1 by CDS upstream and downstream sequences(20bp),and used PCR to amplificate.
[cDNA 1uL, primer1 (3.3uM) 1uL, TaKaRa PrimeSTAR Max DNA Polymerase 2x 10uL,H2O 8uL]
[98℃ 10min; 98℃ 10s, 55℃ 5s, 72℃ 45s,72℃ 7min ]
Thus, We got product1, as shown in the red box of figure 1.(there are 4 different variants, so the products are not only.)
Then, we design 4 primers to get 4 sequences, which are included in the product1, and every primer overlapped 15bp sequences for finally getting amplification product of CDS sequences by PCR.
And we used product1 to amplificate separately to get 4 smaller products(left to right, 310bp, 137bp, 300bp, 231bp), as shown in the red box(product1 1/10 dilution) and green box(product1 1/100 dilution) of figure 2.
[diluted product1 1uL, primer (3.3uM) 1uL, TaKaRa PrimeSTAR Max DNA Polymerase 2x 10uL,H2O 8uL] [98℃ 10min; 98℃ 10s, 55℃ 5s, 72℃ 15s,72℃ 7min ]
After that, we jointed these 4 smaller products.(310bp+137bp, 300bp+231bp, then jointed the 2 products to get the sequences of CDS ), as shown in the figure of question 1, however, the products all flocked together at the sample hole of gel, and no move in the gel, we did twice but no better.
Thank you very much!
Geoff Margison Ekaterina Chesnokova
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