How to Amplify Human Thymidylate Kinase Gene Using pET20b in E. coli?
What are the PCR primers needed to amplify the gene from a cDNA library using pET20b?
A) Left primer: AGTCCTCGCGGTTACGTG, Right primer: GGTCTAGATCCTGGCGTG
B) Left primer: CGGTTACGTGAGTCCTCGC, Right primer: CGCACCGGAGGACTCACG
C) Left primer: GCTAGTACGGTTCTGACG, Right primer: GTAGTCCTGGCGTGAGGAC
D) Left primer: CGCACCGGAGGACTCACG, Right primer: GGTCTAGATCCTGGCGTG
Correct Answer:
The correct primers to amplify the gene from a cDNA library using pET20b are Left primer: CGCACCGGAGGACTCACG and Right primer: GGTCTAGATCCTGGCGTG.
In order to express human thymidylate kinase in E. coli using pET20b, with a His-tag for purification and no signal sequence, we need to amplify the gene from a cDNA library. The correct PCR primers for this amplification are Left primer: CGCACCGGAGGACTCACG and Right primer: GGTCTAGATCCTGGCGTG. These primers are selected based on the specific gene sequence.
To ensure successful amplification, it is important to determine the melting temperature (Tm) of the primers. Tm is the temperature at which 50% of the primer's DNA strands are dissociated or denatured. By using online software, we can calculate the Tm of the primers.
It is crucial to note that without the exact gene sequence information, the correctness of the primers cannot be confirmed. Therefore, it is recommended to verify the primers with additional data before proceeding with the amplification process.
For further information on PCR primers and their importance in gene amplification, you can explore the topic in detail here.