Dear all
I am trying to clone a gene, which only contains only "one intron" in the middle part. I treat the extracted RNA with DNAse and use verso cDNA kit, so the DNA contamination ie very likely not the reason that I get two types (with and without intron) of products when using the cDNA as a template to conduct PCR.
In the PCR, my pairs of primers locating in UTR regions (forward in 5' and reverse in 3'), but when using different pairs of primers for the same cDNA sample, I sometimes get two types, but sometimes only get one type. Theoratically, If the cDNA does contain two types, all pairs of primers should always get two types.
Does anyone know how to explain my result?
Best regards,
The simplest explanation is that your do still have some DNA present. You can easily test this by taking your "RNA" prep and doing PCR directly instead of RT. Another explanation might be inefficient or alternate splicing.
Is there a reason this is important? If you know the correct size product just isolate it and clone it and don't worry about the other product. Or clone and sequence both if you want to.
Are both of the bands of similar size?
If so then there may be a polymorphism in the cdna and in the pcr amplification the different strands are forming a heteroduplex which will run slightly larger than its true size and look like another product. This sometimes happens when there is a lot of pcr product. Sequencing the band will clarify this.
Chia-Hao Chang As you have mentioned your gene has only one intron, the possibility of two bands due to alternative spilling is very less.
Please confirm, out of two bands you are getting whether one is same as size with intron and other one is of size without intron? If answer is Yes then please perform the PCR with your RNA preparation as template with the same primers, if you get the amplification then its due to contamination of genomic DNA.
If answer is No and difference between the two bands is ~ 50 bases or less, then in your PCR with cDNA as template, please extend the final extension cycle till 10 min.
Hope it would be helpful.
Dear all
Thanks for all your response. First, I did use the RNA as a template to do the PCR, and I get nothing. Moreover, the verso cDNA kit contains DNAase. Therefore, I think DNA contamination would not the reason that I get two bands when using cDNA as a template. I coned these two bands and confirmed that the larger one is with intron and the small one is without. This matters to my study.
The other problem is that I tried to use different pairs of primers, and in these primers, forward primers locate in the 5' UTR and the reversed primers are in the second exon, so theoretically, I can always amplify these two bands. However, when using the same cDNA sample as a template and with different pair primers (all PCR settings are the same, ex anealling temperature, No. of cycling ), some pairs amplify more the larger one product, but the others amplify more the smaller one product.
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