Which AAV serotype to use for AAV-hSyn-GFP/Cre injections to the mouse medial septum?

Dear all,

when I analyzed a list of genes mutated in our mouse tumor model in Enrichr, the Users Contributed List 'Sca1+genes' overlapped perfectly, but I cannot find any information about the origin of this gene list.

Can anyone help me? Thank you.

Hello,

I performed an rt-PCR using CRISPR KO cells as I have done numerous times before. I am confident that there has been no error in RNA isolation, cDNA synthesis protocol, or primer usage. However, I did use a brand new cDNA synthesis kit during the cDNA synthesis step. Could this be the cause for my CT values being too high? I cannot think of another reason as I have used these primers with this CRISPR cell line in the past with no issues. Any help will be greatly appreciated!

My supervisor suggested that the bp of PCR products is related to the digestion time. I cannot find anything on the internet to solve my problem. I have a PCR product 500bp. My supervisor implied that I should digest it for longer than the plasmid, why is this?

I tried Gibson Assembly to do cloning recently but all failed.

I used Snapgene to get primers for the amplication of vector and fragment. PCR worked well. Vector is 2.6kb and Fragment is 4.3kb. Notice the fragment (insert) is bigger than the vector.

I used 2X Gibson mix, always 50ng of vector, and tried Vector : Fragment = 3:1 and 1:3, since fragment is bigger than vector. The incubation time I tried 50C 15min, 25min, 60 min. The transformation I tried electrocompentent cells, stabl3 cells, and One shot cells.

I smeared all bacterial onto the selective plate for colony growth.

I could find a few colonies on the plate, but the sequencing results showed they are all original vectors.

Then I further used Dpn I to digest the Gibson reaction products before transformation, and the subsequent sequencing results of the colonies showed that they are the self-circled constructs of vector PCR product.

Anyway, the Gibson assembly did not work. Why? because the fragment is too larger? Do I need to try vector : Fragment=1:1? Do I need to extend the incubation time for Assembly, like 2-3 hours? The overlap of primers is 40 bp.

Thank you so much for your suggestion. I am looking forward to your opinion.

Qinhong Wang

UNC-Chapel Hill

Is it necessary to measure the quantity of single-strand cDNA after RT-PCR and before qPCR? TaqMan Universal PCR Master Mix manual (TaqMan Gene Expression Assay) (at picture) describes the desired quantity of template (1-100 ng) per reaction. So, I should measure the quantity of cDNA after RT-PCR (for instance, on Nanodrop spectrophotometer) and bring this amount to the desired range if needed?

Our lab is using ImageJ to process and compare IHC tumor sections. These sections are of varying areas and have varied signal strengths, and we want to quantifythis to determine if there is a meaningful difference between treatment and non-treatment groups (ex: CD8 cells). How does one account for the area differentials between samples? Would measuring total signal and dividing by total area be accurate? Thanks!

We performed restriction digestion and to analyze the restriction pattern by and received a bad result. What may be the reason behind it

Can anyone please tell me how one would add the amount of dmso necessary for my vehicle controls for cell culture drug treatments? For example, i have four drug concs: 0nm, 200nm, 500nm, 1um. I believe I treat the 0nm group with 1um of dmso, and the 1um group with 1um of the drug. If i treat the 500nm group with 500nm of the drug, do I have to also treat that group with 500nm DMSO, how would I do so?