Good afternoon, you can help me to make five sets of primers for exons 2 to 6 of the G6PD gene from Rattus norvegicus. Hybridization temperature = 60° C, optimum size = 23, 40% GC optimum
Thank you.
a one-liner:
$ curl -s "ftp://ftp.ensembl.org/pub/release-75/gtf/rattus_norvegicus/Rattus_norvegicus.Rnor_5.0.75.gtf.gz" |\
gunzip -c |\
grep -i '"g6pd"' |\
grep -E 'exon_number "[2-6]"' |\
awk -F ' ' '{if($3=="exon") printf("http://www.ensembl.org/das/Rattus_norvegicus.Rnor_5.0.reference/sequence?segment=%s:%d,%d\n",$1,int($4)-50,int($5)+50);}' |\
while read L
do
curl -s "$L" |\
xmllint --xpath '/DASSEQUENCE/SEQUENCE/text()' - |\
awk 'BEGIN{printf("PRIMER_NUM_RETURN=1\nPRIMER_THERMODYNAMIC_PARAMETERS_PATH=/your/path/to/primer3-2.3.5/src/primer3_config/\nPRIMER_OPT_SIZE=20\nPRIMER_OPT_TM=60.0\nPRIMER_OPT_GC_PERCENT=40.0\nSEQUENCE_TEMPLATE=");} {printf("%s",$0);} END{printf("\n=\n");}' |\
/your/path/to/primer3-2.3.5/src/primer3_core
done
output:
PRIMER_OPT_SIZE=20 PRIMER_OPT_TM=60.0 PRIMER_OPT_GC_PERCENT=40.0 PRIMER_NUM_RETURN=1 SEQUENCE_TEMPLATE=gttaatgctcttccattctcctgcctatcaatgagcctttcttccaccagaaaacatcatggcagagcaggtggctttgagccggacccaggtgtgtgggatcctgagggaagagttgtaccagggtgatgccttccaccaagctgatacacacatatttatcatcatgggtgcatcggtaagtctccctttatgccccgagtctaaaagtgcaaggcacttctctct PRIMER_LEFT_NUM_RETURNED=1 PRIMER_RIGHT_NUM_RETURNED=1 PRIMER_INTERNAL_NUM_RETURNED=0 PRIMER_PAIR_NUM_RETURNED=1 PRIMER_PAIR_0_PENALTY=0.285771 PRIMER_LEFT_0_PENALTY=0.178876 PRIMER_RIGHT_0_PENALTY=0.106894 PRIMER_LEFT_0_SEQUENCE=tgtgtgggatcctgagggaa PRIMER_RIGHT_0_SEQUENCE=ctcggggcataaagggagac PRIMER_LEFT_0=92,20 PRIMER_RIGHT_0=201,20 PRIMER_LEFT_0_TM=60.179 PRIMER_RIGHT_0_TM=59.893 PRIMER_LEFT_0_GC_PERCENT=55.000 PRIMER_RIGHT_0_GC_PERCENT=60.000 PRIMER_LEFT_0_SELF_ANY_TH=0.00 PRIMER_RIGHT_0_SELF_ANY_TH=0.00 PRIMER_LEFT_0_SELF_END_TH=0.00 PRIMER_RIGHT_0_SELF_END_TH=0.00 PRIMER_LEFT_0_HAIRPIN_TH=45.78 PRIMER_RIGHT_0_HAIRPIN_TH=0.00 PRIMER_LEFT_0_END_STABILITY=3.9700 PRIMER_RIGHT_0_END_STABILITY=3.3600 PRIMER_PAIR_0_COMPL_ANY_TH=0.00 PRIMER_PAIR_0_COMPL_END_TH=0.00 PRIMER_PAIR_0_PRODUCT_SIZE=110 = PRIMER_OPT_SIZE=20 PRIMER_OPT_TM=60.0 PRIMER_OPT_GC_PERCENT=40.0 PRIMER_NUM_RETURN=1 SEQUENCE_TEMPLATE=gaatgatctaggtcatgtcacctccttctgacatgtttcttctgctgcagggtgacctggccaagaagaagatttatcctaccatctggtaggtaatatctctacactaccccccaatcaaccacaaaatactctcta PRIMER_LEFT_NUM_RETURNED=1 PRIMER_RIGHT_NUM_RETURNED=1 PRIMER_INTERNAL_NUM_RETURNED=0 PRIMER_PAIR_NUM_RETURNED=1 PRIMER_PAIR_0_PENALTY=4.021223 PRIMER_LEFT_0_PENALTY=0.183492 PRIMER_RIGHT_0_PENALTY=3.837731 PRIMER_LEFT_0_SEQUENCE=tgtttcttctgctgcagggt PRIMER_RIGHT_0_SEQUENCE=agtattttgtggttgattggggg PRIMER_LEFT_0=33,20 PRIMER_RIGHT_0=132,23 PRIMER_LEFT_0_TM=59.817 PRIMER_RIGHT_0_TM=59.162 PRIMER_LEFT_0_GC_PERCENT=50.000 PRIMER_RIGHT_0_GC_PERCENT=43.478 PRIMER_LEFT_0_SELF_ANY_TH=22.00 PRIMER_RIGHT_0_SELF_ANY_TH=0.00 PRIMER_LEFT_0_SELF_END_TH=0.00 PRIMER_RIGHT_0_SELF_END_TH=0.00 PRIMER_LEFT_0_HAIRPIN_TH=31.51 PRIMER_RIGHT_0_HAIRPIN_TH=0.00 PRIMER_LEFT_0_END_STABILITY=4.3400 PRIMER_RIGHT_0_END_STABILITY=5.4000 PRIMER_PAIR_0_COMPL_ANY_TH=0.00 PRIMER_PAIR_0_COMPL_END_TH=0.00 PRIMER_PAIR_0_PRODUCT_SIZE=100 = PRIMER_OPT_SIZE=20 PRIMER_OPT_TM=60.0 PRIMER_OPT_GC_PERCENT=40.0 PRIMER_NUM_RETURN=1 SEQUENCE_TEMPLATE=ggccttggctttccctacccagtctgaaaacagctgtactgtaccgtcaggtggctgttccgggatggccttctacccgaagacaccttcattgtaggctatgcccgctcacgactcacagtggatgacatccgcaaacagagtgagcccttctttaaagtgagtatgacattggatcctggagcctttttctgccctttcatctaccc PRIMER_LEFT_NUM_RETURNED=1 PRIMER_RIGHT_NUM_RETURNED=1 PRIMER_INTERNAL_NUM_RETURNED=0 PRIMER_PAIR_NUM_RETURNED=1 PRIMER_PAIR_0_PENALTY=0.222430 PRIMER_LEFT_0_PENALTY=0.034089 PRIMER_RIGHT_0_PENALTY=0.188342 PRIMER_LEFT_0_SEQUENCE=ccttggctttccctacccag PRIMER_RIGHT_0_SEQUENCE=aagggcagaaaaaggctcca PRIMER_LEFT_0=2,20 PRIMER_RIGHT_0=198,20 PRIMER_LEFT_0_TM=60.034 PRIMER_RIGHT_0_TM=59.812 PRIMER_LEFT_0_GC_PERCENT=60.000 PRIMER_RIGHT_0_GC_PERCENT=50.000 PRIMER_LEFT_0_SELF_ANY_TH=0.00 PRIMER_RIGHT_0_SELF_ANY_TH=0.00 PRIMER_LEFT_0_SELF_END_TH=0.00 PRIMER_RIGHT_0_SELF_END_TH=0.00 PRIMER_LEFT_0_HAIRPIN_TH=0.00 PRIMER_RIGHT_0_HAIRPIN_TH=0.00 PRIMER_LEFT_0_END_STABILITY=4.4500 PRIMER_RIGHT_0_END_STABILITY=3.8600 PRIMER_PAIR_0_COMPL_ANY_TH=17.01 PRIMER_PAIR_0_COMPL_END_TH=8.74 PRIMER_PAIR_0_PRODUCT_SIZE=197 = PRIMER_OPT_SIZE=20 PRIMER_OPT_TM=60.0 PRIMER_OPT_GC_PERCENT=40.0 PRIMER_NUM_RETURN=1 SEQUENCE_TEMPLATE=aagtgtagactcttgagaaaagtagttggcattgttctgtgtatctccaggtcactccagaagaaagacccaagctagaggagttctttgcccgtaactcctatgtagctggccagtatgatgatccagcctcctacaagcacctcaacagccacatgaatgccctgcaccagggaatgcaggccaaccgtctgttctacctggccttgccccccactgtctatgaagcagtcaccaagaacattcaagagatctgcatgagtcagacgtaaggcctgtctttccccactcctgtctgtttgctcttgtttctactac PRIMER_LEFT_NUM_RETURNED=1 PRIMER_RIGHT_NUM_RETURNED=1 PRIMER_INTERNAL_NUM_RETURNED=0 PRIMER_PAIR_NUM_RETURNED=1 PRIMER_PAIR_0_PENALTY=0.217287 PRIMER_LEFT_0_PENALTY=0.107945 PRIMER_RIGHT_0_PENALTY=0.109342 PRIMER_LEFT_0_SEQUENCE=cagcctcctacaagcacctc PRIMER_RIGHT_0_SEQUENCE=agcaaacagacaggagtggg PRIMER_LEFT_0=126,20 PRIMER_RIGHT_0=303,20 PRIMER_LEFT_0_TM=60.108 PRIMER_RIGHT_0_TM=59.891 PRIMER_LEFT_0_GC_PERCENT=60.000 PRIMER_RIGHT_0_GC_PERCENT=55.000 PRIMER_LEFT_0_SELF_ANY_TH=0.00 PRIMER_RIGHT_0_SELF_ANY_TH=0.00 PRIMER_LEFT_0_SELF_END_TH=0.00 PRIMER_RIGHT_0_SELF_END_TH=0.00 PRIMER_LEFT_0_HAIRPIN_TH=0.00 PRIMER_RIGHT_0_HAIRPIN_TH=0.00 PRIMER_LEFT_0_END_STABILITY=3.8500 PRIMER_RIGHT_0_END_STABILITY=4.6100 PRIMER_PAIR_0_COMPL_ANY_TH=0.00 PRIMER_PAIR_0_COMPL_END_TH=0.00 PRIMER_PAIR_0_PRODUCT_SIZE=178 = PRIMER_OPT_SIZE=20 PRIMER_OPT_TM=60.0 PRIMER_OPT_GC_PERCENT=40.0 PRIMER_NUM_RETURN=1 SEQUENCE_TEMPLATE=ctattcctgggaaagatttgagaaataacatgattgggggctctaagcagaggctggaaccgcatcatagtggagaagcccttcgggagagacctgcagagctccaatcaactgtcgaaccacatctcctctctgtttcgtgaggaccagatctaccgcattgaccactacctgggcaaagagatggtccagaacctcatggtgctgaggtgcacgggggatggggtggaaaaggaccagagcaagggtggcaatgtga PRIMER_LEFT_NUM_RETURNED=1 PRIMER_RIGHT_NUM_RETURNED=1 PRIMER_INTERNAL_NUM_RETURNED=0 PRIMER_PAIR_NUM_RETURNED=1 PRIMER_PAIR_0_PENALTY=0.071813 PRIMER_LEFT_0_PENALTY=0.033503 PRIMER_RIGHT_0_PENALTY=0.038310 PRIMER_LEFT_0_SEQUENCE=tgaggaccagatctaccgca PRIMER_RIGHT_0_SEQUENCE=ctctggtccttttccacccc PRIMER_LEFT_0=140,20 PRIMER_RIGHT_0=241,20 PRIMER_LEFT_0_TM=60.034 PRIMER_RIGHT_0_TM=59.962 PRIMER_LEFT_0_GC_PERCENT=55.000 PRIMER_RIGHT_0_GC_PERCENT=60.000 PRIMER_LEFT_0_SELF_ANY_TH=15.47 PRIMER_RIGHT_0_SELF_ANY_TH=0.00 PRIMER_LEFT_0_SELF_END_TH=14.04 PRIMER_RIGHT_0_SELF_END_TH=0.00 PRIMER_LEFT_0_HAIRPIN_TH=0.00 PRIMER_RIGHT_0_HAIRPIN_TH=0.00 PRIMER_LEFT_0_END_STABILITY=5.6900 PRIMER_RIGHT_0_END_STABILITY=4.9500 PRIMER_PAIR_0_COMPL_ANY_TH=17.76 PRIMER_PAIR_0_COMPL_END_TH=17.33 PRIMER_PAIR_0_PRODUCT_SIZE=102 =
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version 2.3.6
Since this is in the "Question" category, we'll assume that you meant to say "Can you help me". Otherwise it sounds like a demand :)
Have you tried making them yourself with a program like Primer3, Primer3Plus, or PrimerQuest (there are a good number of these)?
Yes, I have tried to use Primer3web but I do not go completely. I just goes full exon 2.
I'm Panamanian, I don't speak English very well
So were you able to retrieve primer sequences for exon 2 or not (sorry, it's unclear)? If so, just do the remaining exons, then. If not, did you receive an error and, if so, what was it?