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2024-03-28T22:46:51Z
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https://wiki.houptlab.org/wiki/PCR
PCR
2024-02-14T15:11:23Z
<p>Houpt: notes on PCR</p>
<hr />
<div><br />
==Primers==<br />
<br />
To dilute primers to a stock of 100 µM, add 10 µl of water for each nmol of lyophilized primer. For example, if there are 38.2 nmol of primer a 100 µM primer stock is created by adding 382 µl of water.<br />
<br />
Dilute 10 µM working stock aliquots from the 100µM stock 10-fold, e.g. 10 µl of 100µM and 90 µl of water.</div>
Houpt
https://wiki.houptlab.org/wiki/JCF_Excitatory_DREADD_Functional_Validation
JCF Excitatory DREADD Functional Validation
2024-01-25T21:05:17Z
<p>Jcote: /* Agenda */</p>
<hr />
<div>To validate FosCreER x DREADD mice by testing if active cellular populations after LiCl exposure can be reactivated with CNO administration to induce a taste aversion. <br />
<br />
<br />
<br />
CNO ordering information / lot numbers<br />
<br />
CNO preparation (e.g. vehicle, how its dissolved)<br />
<br />
4-OHT preparation: Dissolved in pharmaceutical grade sesame oil 5mg/ml shaking o/n 55c<br />
<br />
{{Infobox experiment|align=right<br />
| Name = Clozapine-N-Oxide Taste Aversion<br />
| project = DREADDs and Magentic Field<br />
| code = JCF<br />
| protocol = 202200000006<br />
| funding = Chair's Fund<br />
| investigators = Cote, Houpt<br />
| species = mice<br />
| date = 2024-1-28<br />
| type = CTA<br />
| drugs = NaCl, CNO<br />
| artifacts = <br />
| data = https://houptlab.org/bartab/expt/?id=JCF}}<br />
<br />
<br />
<br />
==Data== <br />
<br />
'''[https://houptlab.org/bartab/expt/?id=JCF Raw Data Link]'''<br />
<br />
==People== <br />
<br />
Jason Cote<br />
<br />
==Agenda==<br />
<br />
Mice In House Colony FosCreER X hM3Dq (n=?? male DOB +/-, n=?? female DOB +/-)<br />
<br />
Drug Preparation: Isotonic LiCl (0.15M,20ml/kg, i.p) followed by 4-OHT prepared at 5mg/ml (50mg/kg,10ml/kg, i.p) or Sesame oil vehicle (10ml/kg i.p) after 10 min. <br />
<br />
Pre Treatment (1/28/24) 2 weeks before starting water restriction<br />
4 groups:<br />
<br />
1. Isotonic LiCl and 4-OHT, N=12: Excitatory DREADDs are expected to be expressed<br />
<br />
2. Isotonic Saline and sesame oil, N=11: No DREADDs are expected to be expressed<br />
<br />
<br />
'''Day 0: Water removed''' (**2024-7-19)<br />
<br />
<br />
'''Days 1 - 11: Water Restriction''' (2023-9-13 - 2023-9-23)<br />
<br />
Weigh mice daily. Ad lib food. Water access on consecutive days at noon: 3h, 3h, 3h, 1h, 1h, 1h, 30min, 30 min, 10 min, 10 min, 10 min. <br />
<br />
<br />
<br />
Mice given water bottle and empty bottle, alternating position. <br />
<br />
In the afternoon (~4pm), mice will be given supplemental access to water for 30 min.<br />
<br />
'''Day 12: Conditioning Day''' (2024-2-28)<br />
<br />
Mice are given 10-min access to 0.125% saccharin solution, then injected with CNO (3mg/kg,10ml/kg, i.p) after 15 min <br />
<br />
Groups: <br />
* LTC: Exp group (Predicted to form taste aversion)<br />
* VVC: Control (expect to not form taste aversion) <br />
<br />
<br />
* JCF01-04: ???<br />
* JCF05-08: ???<br />
* JCF09-12: ???<br />
* JCF13-16: ???<br />
<br />
After injection, ad lib water returned overnight.<br />
<br />
'''Day 13 - 24: Preference Tests''' (2023-9-25 - 2023-10-6 )<br />
<br />
CTA acquisition and extinction was measured with 24-h, 2-bottle preference tests. Mice were given 24-h free access to both 0.125% saccharin and water bottles, alternating position for 7 days.<br />
<br />
<br />
[[Category:Taste Aversion]]<br />
[[Category:CTA Experiments]]<br />
[[Category:Magnet Experiments]]</div>
Jcote
https://wiki.houptlab.org/wiki/Anorexia_anx/anx_mutant_mice
Anorexia anx/anx mutant mice
2024-01-16T16:33:43Z
<p>Houpt: added some orders</p>
<hr />
<div><br />
Anorexic anx/anx mice [https://www.jax.org/strain/000624 Jackson Lab strain 000624].<br />
<br />
Mutation mapped to a 0.2 cM interval residing between the markers D2Mit133 and Jojo5 chromosome 2 (Chr 2: bp 118, 889, 896–120, 175, 1081) (Lindfors et al., 2011).<br />
<br />
<br />
== Possible genes affected ==<br />
<br />
Lindsfor 2011: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1) <br />
<br />
Kim 2017: tyrosine kinase receptor Tyro3 which they conclude is not the anx-mutation but a strain specific modifier of anx-phenotypes <br />
<br />
== Abnormalities Table ==<br />
<br />
== Tyro3 Genotyping ==<br />
<br />
TYRO3 protein tyrosine kinase 3 (Tyro3) mutation in anx/anx eliminatees a NlaIV restriction site.<br />
<br />
* primers 5′-GATGGCGCTGAGGCGGAGCATG and 5′-CGCGGC- CGGAGGTCTGGCAG ; annealing temperature 72C<br />
<br />
* subsequent digestion with NlaIV <br />
<br />
* wildtype -> 175 bp fragment, anx/anx -> 250 bp product<br />
<br />
[[File:anx_pcr_kim_2017.png|800px|thumb]]<br />
<br />
<br />
<br />
== Genotyping 1998 mice ==<br />
<br />
* 2024-1-17 Tyro3 primers ordered from FSU BIO Core <br />
<br />
* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product%20Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product%20Information 50bp ladder]</div>
Houpt
https://wiki.houptlab.org/wiki/JG_Selenate_and_LiCl-induced_pMAPK_and_c-Fos
JG Selenate and LiCl-induced pMAPK and c-Fos
2024-01-12T17:58:08Z
<p>Houpt: added categories</p>
<hr />
<div>Expt Code: JG<br />
<br />
TODO: get dates and number of rats, DOBs, experiment info box<br />
<br />
IACAUC Protocol: 1113<br />
<br />
# Animals<br />
<br />
* Adult male Sprague Dawley rats (n=12)<br />
* weight 300 - 500g<br />
* Source: Charles River<br />
* DOB:<br />
<br />
<br />
# Procedure (rev 2013-7-24)<br />
<br />
Weight rats<br />
<br />
## Schedule: <br />
<br />
(Stagger pairs of rats by 20 min to allow time for perfusion)<br />
<br />
at 0h, inject with either 1 ml/kg vehicle (0.15 M NaCl) or 0.5 mg/kg Na2SeO4 <br />
<br />
at 1 h, inject with either 12 ml/kg NaCl or 12 ml/kg LiCl (0.15 M)<br />
<br />
at 2 h, overdose with Sleepaway and perfuse<br />
<br />
Dissect brains, postfix for 2 h in 4%PFA, rinse with 0.1 M PB, cyroprotect in 30% sucrose<br />
<br />
# Immunohistochemistry<br />
Process brains for immunohistochemistry with anti-cFos and anti-pMAPK:<br />
<br />
From Kwon and Houpt:<br />
<br />
anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) rabbit mAb (#4370, 1:500, Cell Signaling Technology)<br />
<br />
From Kimbrough TORC-1 paper:<br />
<br />
anti-c-Fos goat polyclonal antibodies (#SC-52-G, 1:10,000 Santa Cruz Biotechnology)<br />
<br />
<br />
[[Category:CTA_Experiments]]<br />
[[Category:Taste_Aversion]]</div>
Houpt