Paramecia in situ PCR?

11 November 2015 1 3K Report

I am attempting in situ PCR with paramecium. Has anyone tried in situ PCR in a similar organism with a pellicle? I trying to determine the parameters for fixation and protease treatment. Any help welcomed.

Rafik Karaman

Dear David,

The attached paper might be helpful.

Rafik

Badges
Science method

More David A Johnson's questions See All

Can I use a Taq mastermix for A-tailing?

I have gel-purified PCR product from a Q5 high-fidelity amplification to which I want to add the A tails for TOPO-TA cloning. The protocols I find all say to use Taq polymerase and dATP, but can I...

08 September 2019 6,894 3 View

For inverse PCR, is it OK if my restriction enzyme cleaves my known DNA once?

I am running inverse PCR to try to sequence the region flanking a novel gene. My question is whether or not it is OK to use a restriction enzyme that cleaves my known sequence at one site. That...

06 July 2019 5,486 4 View

How do I design inverse PCR primers for environmental sample with multiple sequences?

I have an environmental genomic DNA sample that includes three different newly-discovered 134 nt novel sequences. I want to do inverse PCR (using restriction enzymes and ligation) to begin...

04 May 2016 4,215 1 View

Can I use PCR reaction run with normal primers as template source for inverse PCR reaction?

I will be running inverse PCR to sequence the segments adjacent to my target segment. One of the template sources will be very difficult obtain again. I first ran the non-inverse PCR to discover...

03 April 2016 2,164 4 View

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

I can't see the ssDNA band after performing asymmetric PCR. Is there any way to do this?

After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...

08 August 2024 1,668 3 View

How to normalize and take the significance of the MTT OD values with 3 replicates for the same cell-line?

Hi, I have a question about normalizing the MTT OD values for doing the statistical analysis. So, if we have 3 different plates and we call them 3 different replicates, so, first we would...

07 August 2024 8,106 4 View

Why activated CAR-Jurkat cell could not kill targets?

Previously when I co-coluture anti-CD19(FMC63) CAR-Jurkat with Raji with E:T=5:1, Jurkat can eliminate Raji in 24h. However, when I test another CAR construct, although I can dectect totally CD69...

06 August 2024 641 2 View

Why does my protein refolded to beta sheet during thermal denaturation analysis?

Hi! So i attempted to understand a novel protein behavior towards heat application by analyzing its secondary structure change. I subjected the protein to a thermal denaturation analysis using...

06 August 2024 1,989 3 View

How much total RNA concentration to be extracted from sorted plasma cells from bone marrow of C57BL/6 mice for RT-PCR ?

i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...

05 August 2024 8,835 1 View

Weak DAPI staining after immunohistochemistry - how to improve?

After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without...

05 August 2024 9,637 2 View

Low-yield gel extraction problem?

I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...

05 August 2024 9,798 3 View

How to isolate lymphocytes from mouse spleen?

I have tried several times to isolate lymphocytes from mouse spleen, but all attempts have been unsuccessful. I tried most available protocols. I used different dissociation media (HBSS with Ca...

04 August 2024 9,913 7 View

Does anyone have issues using Prepman Ultra reagent for MicroSeq ID bacterial, fungal and yeast sample preparation?

I have been attempting to extract DNA from Bacterial, Fungal and Yeast banked samples (>1e7 cells) using Prepman Ultra reagent and I seem to be struggling to obtain a sequence. Although the...

01 August 2024 2,079 0 View