Özlem Gönülkırmaz

Hebrew University of Jerusalem | HUJI · Department of Ecology, Evolution and Behavior

I am planning to work with two guides for my next CRISPR injections to be able to have a long deletion. I am wondering about the strategies. For instance, I have verified gRNAs as in vitro and I aim to design the second gRNAs nearby like in 20-30 bp distance but finding two good gRNAs this much close to each other and in opposite direction is difficult in some regions. Do you think it would affect the efficiency if one of them has a high possible off-target score while the other one does not have any? How can I make a good combination of two guides?

I am trying to understand the "self complementarity" term in PCR primer design. As I understand, it is desired to have the score less than 4 matches for the 3' end to avoid heterodimer structure. But I didn't understand the logic of the score for self complementarity. Based on the NCBI primer blast, when I design the primers, it gives two parameters: "Self complementarity" and "Self 3' complementarity". What is the desired range for the first one to be able to design specific PCR primers without dimerization and why?

The size of the eggs are huge in comparison with some other insects and around 3.0-3.5 mm in lenght. So when I tried to stain with DAPI, I had autofluorescent in every part of the embryo and it is not penetrating in deeper parts of the embryo as can be seen on confocal. I am trying to see nuclei at different stages. Does anyone has any suggestion with different dye or technique?

I need to check nuclei in the eggs but the egg size is quite big for our microscopy so I couldn't see any nucleus stained.

I'm using Biopython codes to parse 4 whole genome fasq files. One of them is around 11 GB and working on them is difficult and so slow. I need to compare these 4 genomes variability for specific gene regions and also need to search for specific sequence pieces (guide sequences for Crispr). But I don't know how to find and read sequences in this kind of data. Could someone please help me?

I just know their record numbers and total sequence lenghts with this code:

from Bio.SeqIO.QualityIO import FastqGeneralIterator

count = 0

total_len = 0

with open("document.fastq") as in_handle:

for title, seq, qual in FastqGeneralIterator(in_handle):

count += 1

total_len += len(seq)

print("%i records with total sequence length %i" % (count, total_len))

I have done two-way repeated measure ANOVA for my thesis data but I could not understand how F and P values can be calculated with df (degrees of freedom). I need help.. Thanks.

Double staining is necessary or not? I know, lipofuscin is consist of autofluorecence protein-lipid content so Sudan Black B can stain and it can be showed by using fluorescent microscopy. But also, I read an article about cresyl violet staining method is applicable and there were light microscopy images of lipofuscin with this staining. Besides, in another article they were stained using a combined Alcian Blue and Periodic Acid-Schiff technique (AB PAS). I am confused with so many methods and not sure which one is the best for showing accumulation on rat brain tissue by using light microscopy. Maybe, only light microscopy is not enough to detect. Could someone who used this techniques give some information to clear up for me?

I tested some blood serum parameters like HDL, LDL, trigyceride etc. and leptin in albino wistar rats. When I look at the literature I could not find a direct answer about right timing of blood collection. If I applied sample collection in the same way, same time, same row for all animals, can eating of rats before blood collection still create a huge individual variance differences and avoid to have significant data for treatment? Please help me, I am completely confused.

Is there any specific protocol for this kind of staining?

Please help me if you use this method in your researches. I read some articles about there are difficulties to see lipofuscins with this staining. After slicing the total brain what procedure should I use?