Animal Development Project Lab - Target Genes

Team Ecto: dsh-1, hmr-1, mig-14, ref-1

Team Meso: egl-15, lit-1, mom-1, prkl-1

Team Endo: dlg-1, gsk-3, lin-45, sos-1

Team NC: elt-1, lin-3, unc-37, vang-1

Progress toward getting your RNAi DNA clone in the RNAi bacteria:

Previous updates: 10-18-13 update, 10-21-13 update, 10-26-13 update, 11-6-13 update, 11-13-13 update, 11-20-13 update

Use your primer sequences to determine the amplicon size, and part of the gene being amplified.

Note: the following primers are now correct for the named genes: egl-15, elt-1, hmr-1, lin-45, mig-14, ref-1

Oligo name Sequence Oligo name Sequence
dlg-1_F TGAAGCATGAGGTTGCAGTC dlg-1_R AACCAGATCAATGACGGATG
dsh-1_F CACATCAGTATCCCGACAACA dsh-1_R CAAAGCTGGTCTCATTGACTTG
egl-15_F2 GGCCATACATTTGCTCACTG egl-15_R2 GGATCCGAATCAACCTCGTA
elt-1_F2 TCCAACGACACAACTCAACA elt-1_R2 ATTGTTGGCTCCTCCTGATG
gsk-3_F AAGCAGCGACAACAAATTCC gsk-3_R TTGTTCACGAGTCGGTGTTC
hmr-1_F2 GAGCCACAGAACACCGAGTC hmr-1_R2 GGATTACTCGGATCGTGAGC
lin-3_F TCATATGTGCCCACCAGAAG lin-3_R ACTTCAACACGTGGCACTGA
lin-45_F2 AACTCTTTGGGATCGCATCA lin-45_R2 TGTTGAACTGTTGCCTCATCA
lit-1_F GTATACGGGAGCCGTGGATA lit-1_R TCTTATCCGGATCGAAGTGC
mig-14_F2 ACGCGGAATCCAGCTCTAC mig-14_R2 ATCCATCACGAGCTTCATCC
mom-1_F TTTCAACAACGGTTCGTGTC mom-1_R TTTCCAGATTGAAGGCAACC
prkl-1_F AACCCGTGTGCTTGAAATGT prkl-1_R TGACATGAGGATGACGTGGT
ref-1_F2 TGTCTTAAACCGTGCAGTCG ref-1_R2 AGATTCCGATGTCGATGGAG
sos-1_F TGGCTGTTCTGGTGAAAGTG sos-1_R CACCTTGTTTCGGTTGTTGA
unc-37_F AGCTTTCAAAACCGCATCAC unc-37_R CCCGATGAGGCAACACTATT
vang-1_F CCTCGAGCTTCGTCGTCTAC vang-1_R CTTCCTGCATCACTTCAGCA

F = forward; R = reverse

Go to Reverse e-PCR in new window or tab

Enter the information as shown below. All of the sequences being amplified (the 'amplicon') are in the size range
of 500 bp or less, so the numbers shown will find the correct sequence (this allows a size from 0-1000 bp).

Your results will look something like this:

Next, use Wormbase e-PCR to get what part of the gene is being amplified by the reaction.
(Although you could also use Wormbase e-PCR to figure out the size, NCBI e-PCR shows that number more clearly/easily.)

On the Wormbase home page, find e-PCR under the Tools menu.

On the Wormbase e-PCR page, follow the instructions shown, and then 'search.'

A successful search will show a range of sequence amplified, with a link that will take you to a map of the region.

The first view will fill the page with your amplicon (the sequence being small relative to the entire gene,
it will be very close up. You can select a zoom out showing more sequence to get a better view of the whole
gene. A good place to start is 5 kbp (5 kilo base pairs, 5000 bp); you may need to zoom out more if it is a large C. elegans gene.

In this view showing 5000 bp, the entire coding region of the ceh-20 gene is shown, revealing the amplicon (black) includes most of exon 3.