Hi,
I want to check gene sequence that is a large size (3.7kb). This gene is located in MCS of pQE9 vector (Qiagen).
How many primers that i should design for check full gene ?
Do i need to sequence double strand (using both forward primer and reverse primer)
Thank you for your suggestion
If you need to know the gene identity of the insert, one-way primer gene sequencing is enough. For gene seq >1kb2kb (by convention good sequencing gives ~1000bp nucleotide seq.).
If you do not know about your insert: Use for and rev primer of the vector ... with two 1kb nucleotide sequences from the sequencing reaction, a BLAST will give reliable results.
Mostly the situation is the following: You aim at a special gene, amplify by PCR, clone into a vector, transform a bacterium, do plasmid prep, send to sequening lab. In this case: make sure you used proofreading taq in your initial PCR to get a reliable sequence. Today its state of the art even to be sure that a SNP is inherited , not a taq artifact.
Back to sequencing: 3,7 kb insert needs minimal 4 sequencing reactions.
1st: use for primer from vector
2nd: use rev primer from vector
3rd: You know your candidate gene and its sequence and want to save time by preparing also the internal ones ca 900 nt away from the insertion site? You will save time and postal charges, because you send all 4 sequencing reactions once? DON'T DO! Sometimes the sequencing is not that effective, stops at 700 nt and you have a gap in your nucleotide sequence. Sometimes there are SNPs ... Once my 5'end of my designed primer was a SNP and therefore the primer did not work. You will spend some sequencing reactions and time in troubleshooting.
So 3rd step is: send sequencing reactions from 1st and 2nd to the sequencing lab, wait for your sequence data and use this sequence data to create your internal primers (ca 100 bp away from the end of your reliable sequening data). Never act on asumptions ... do not asume to know your sequence.
regards
I agree with Jörg Vogt. According to Amanda comment; it good in case you have conserve sequence in your gene. However, if your gene not, for example vaccine candidate gene may not work.
Good luck for all.
I used at least 4 primers for 2kb gene to cover in both directions because our sequencer usually yields 500-700nt with decent Q-score per sample.
- Badges
- Science method