Started a PCR with Terebratalia wnta, wnt2; and Meara ago a, ago b, ago c, bru a, bru b, mago, mex3, orb2.
Put colonies to grow.
| Colony | T7 |
| Ago a c1 | BV235 |
| Ago a c2 | BV236 |
| Ago a c3 | BV237 |
| Ago b c1 | BV238 |
| Ago b c2 | BV239 |
| Ago b c4 | BV240 |
| Bru a c1 | BV241 |
| Bru a c2 | BV242 |
| Bru a c3 | BV243 |
| Mago c1 | BV244 |
| Mago c2 | BV245 |
| Mago c3 | BV246 |
| Orb2 c1 | BV247 |
Initial cloning of Meara RNA binding proteins. Selection: ago a, ago b, ago c, boule, bru a, bru b, gus a, mago, mex3, orb2, stau.
Only 3 genes had visible bands: boule, gus a, and stau:
Ligated these overnight.
Transformation and plated the genes.
Colony PCR with 6 colonies from each:
Put colonies to grow.
| boule c1 | |
| boule c2 | |
| boule c3 | |
| gus a c1 | |
| gus a c2 | |
| gus a c3 | |
| stau c1 | BV232 |
| stau c2 | BV233 |
| stau c3 | BV234 |
Extracted plasmid and did sequencing PCR for all using T7 primer. Dropped tubes into sequencing facility.
Checking sequences and they look good, but there is a problem! Minipreps BV226, 227, 228 aligned with gus a and not with boule!!! Check why…
Set a PCR to clone: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b
Bands with expected lengths. Purified DNA, set ligation reaction for 1h45 at RT, heatshock transformation and plated colonies with Xgal.
Plates were wetty, so colonies did not spread well… some were forming strings; but enough to pick. Picked white colonies for colony PCR:
Sequencing PCR and dropped in the SeqLab.
| BV75 | Membranipora membranacea | Mex3 | T7 |
| BV76 | Membranipora membranacea | Fez | T7 |
| BV77 | Membranipora membranacea | Fez | T7 |
| BV78 | Membranipora membranacea | Fez | T7 |
| BV79 | Membranipora membranacea | PL10 | T7 |
| BV80 | Membranipora membranacea | PL10 | T7 |
| BV81 | Membranipora membranacea | PL10 | T7 |
| BV82 | Membranipora membranacea | FoxG | T7 |
| BV83 | Membranipora membranacea | FoxG | T7 |
| BV84 | Membranipora membranacea | FoxG | T7 |
| BV85 | Membranipora membranacea | Syt1a | T7 |
| BV86 | Membranipora membranacea | Syt1a | T7 |
| BV87 | Membranipora membranacea | Syt1a | T7 |
| BV88 | Membranipora membranacea | Syt1b | T7 |
| BV89 | Membranipora membranacea | Syt1b | T7 |
| BV90 | Membranipora membranacea | Syt1b | T7 |
I picked 19 colonies from the previous PCRs of Mm DDX4 and Mm MEX3. Trying to find a positive colony, specially for Vasa.
No positive colonies for MEX3. For Vasa the length was not the same as expected, but I will sequence 6, 7, 10, 15, 17 to check.
Cloning newly collected genes of Membranipora and Priapulus. Selected genes and primers:
Membranipora:
Mm PIWIL1 R1 CCGTTGTTGACCTGATGCCACTTTCG Mm PIWIL1 R2 ACGATGTGATGCTCTCCCTGCTCCATTACC Mm DDX4 R1 TCTCTGTGTCTTGTCTGGCATACCCATCG Mm DDX4 R2 CAGTCACCTTTCTCGGGTTTGGACACTCTC Mm AGO2 F CTTCGCACCTCAGAGGGTAG Mm AGO2 R CTGGCCCTGAAAGCTACAAG Mm MEX3 F CCAGTGAGAGGAGCTGAACC Mm MEX3 R CATCACCACCACACAAGAGG Mm MAGOH F TCCGCCATTCTTTCTCTTTG Mm MAGOH R TGGTTGAATGCAAGATGGAA
Priapulus:
Pc PiwiA F GGGTCTTATTCTTTGTTTGC Pc PiwiA R TTCACGACACCATCGCAG Pc PiwiB R1 TGCGATACTGCCGACCATCTTCTTGTCC Pc PiwiB R2 ACGGGATGTGATGGGCTGTAACCTTTCG Pc Tudor R1 TCCACAAAATAGTAGGCAAGGCAAGGGCTC Pc Tudor R2 ACGCAACAGATGAACACCTCTACGGCTTGG Pc Mael F1 CGTGAAAGAGAGCTGGTCCAGTCAGAATGG Pc Mael F2 CACCGCTTCATAGCTCCAGGTGAAATCC Pc Mael R1 GCGGTGAAATTCCCTCATAATGCCCTTCTC Pc Mael R2 TGGACCAGCTCTCTTTCACGCTTATTTCG Pc Pum F1 ATGAACGCAGTGCCTTGGAACGACTCTCAC Pc Pum F2 GCAGGACGATGCTATGGTCGGCTATTTC
Regular and first round of RACE with the above primers.
Second round of RACE. Run a gel with only 5 µL of each to identify bands to be cut from RACE and direct purify the products of the regular PCR. Ran another gel with the remaining product to cut the bands*. Extracted promising bands and set the ligation reaction at 22:30.
Heat-shock transformation and plating. Also ligation for remaining bands (Mm PIWIL1, Pc Tudor 2,3,4, Pc PUM).
Colony PCR.
Colony PCR for the remaining bands and repetition for some that there was no positive colonies.
Plasmid extraction using 1.5 mL of miniprep culture, sequencing reaction, and dropped at the sequencing facility.
Sequences BV25-BV64 returned.
| BV25 | Membranipora membranacea | Argonaute2 | T7 | ok | + | SP6 |
| BV26 | Membranipora membranacea | Argonaute2 | T7 | ok | + | SP6 |
| BV27 | Membranipora membranacea | Mago Nashi | T7 | ok | – | T7 |
| BV28 | Membranipora membranacea | Dicer1 | T7 | ok | + | SP6 |
| BV29 | Membranipora membranacea | Dicer1 | T7 | ok | + | SP6 |
| BV30 | Membranipora membranacea | Germ Cell Less R2 | T7 | ok | – | T7 |
| BV31 | Membranipora membranacea | PiwiL1 R2 b2 | T7 | ok | – | T7 |
| BV32 | Membranipora membranacea | PiwiL1 R2 b2 | T7 | ok | + | SP6 |
| BV33 | Priapulus caudatus | PiwiB R2 b2 | T7 | ok | – | T7 |
| BV34 | Priapulus caudatus | Tudor R2 b1 | T7 | ok | – | T7 |
| BV35 | Priapulus caudatus | Maelstrom F2 | T7 | ok | + | SP6 |
| BV36 | Priapulus caudatus | Maelstrom F2 | T7 | ok | – | T7 |
| BV37 | Priapulus caudatus | Tudor R2 b2 | T7 | ok | – | T7 |
| BV38 | Priapulus caudatus | Tudor R2 b2 | T7 | ok | – | T7 |
| BV39 | Priapulus caudatus | Tudor R2 b3 | T7 | ok | – | T7 |
| BV40 | Priapulus caudatus | Tudor R2 b3 | T7 | ok | + | SP6 |
| BV41 | Priapulus caudatus | Tudor R2 b4 | T7 | ok | + | SP6 |
| BV42 | Priapulus caudatus | Tudor R2 b4 | T7 | ok | + | SP6 |
| BV43 | Priapulus caudatus | Pumilio F2 | T7 | ok | + | SP6 |
| BV44 | Priapulus caudatus | Pumilio F2 | T7 | ok | – | T7 |
| BV45 | Membranipora membranacea | Argonaute2 | SP6 | ok | – | SP6 |
| BV46 | Membranipora membranacea | Argonaute2 | SP6 | ok | – | SP6 |
| BV47 | Membranipora membranacea | Mago Nashi | SP6 | ok | + | T7 |
| BV48 | Membranipora membranacea | Dicer1 | SP6 | ok | – | SP6 |
| BV49 | Membranipora membranacea | Dicer1 | SP6 | ok | – | SP6 |
| BV50 | Membranipora membranacea | Germ Cell Less R2 | SP6 | ok | + | T7 |
| BV51 | Membranipora membranacea | PiwiL1 R2 b2 | SP6 | ok | + | T7 |
| BV52 | Membranipora membranacea | PiwiL1 R2 b2 | SP6 | ok | – | SP6 |
| BV53 | Priapulus caudatus | PiwiB R2 b2 | SP6 | ok | + | T7 |
| BV54 | Priapulus caudatus | Tudor R2 b1 | SP6 | ok | + | T7 |
| BV55 | Priapulus caudatus | Maelstrom F2 | SP6 | ok | – | SP6 |
| BV56 | Priapulus caudatus | Maelstrom F2 | SP6 | ok | + | T7 |
| BV57 | Priapulus caudatus | Tudor R2 b2 | SP6 | ok | + | T7 |
| BV58 | Priapulus caudatus | Tudor R2 b2 | SP6 | ok | + | T7 |
| BV59 | Priapulus caudatus | Tudor R2 b3 | SP6 | ok | + | T7 |
| BV60 | Priapulus caudatus | Tudor R2 b3 | SP6 | ok | – | SP6 |
| BV61 | Priapulus caudatus | Tudor R2 b4 | SP6 | ok | – | SP6 |
| BV62 | Priapulus caudatus | Tudor R2 b4 | SP6 | ok | – | SP6 |
| BV63 | Priapulus caudatus | Pumilio F2 | SP6 | ok | – | SP6 |
| BV64 | Priapulus caudatus | Pumilio F2 | SP6 | ok | + | T7 |