A predetermined methodology for carrying out an experiment
1
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0answers
34 views
Low RNA yield, low 260/230 ratio
I extracted total RNA from animal tissue using the Qiagen RNeasy kit, however my RNA yield was extremely low and the 260/230 ratio was around 0.3. This is the protocol I followed:
The animal ...
1
vote
0answers
23 views
Removing bacterial contaminants from resin
We have a few Strep-tactin columns that had some growth in them and we would like to regenerate the columns back since the resin is quite expensive. Basic goal: remove the brown stuff.
So far, I've ...
1
vote
1answer
33 views
In vitro enzyme production
I need to express a protein in vitro but I don't know where to start. I will likely do a T7 transcription protocol but for translation I am not sure what to do. Are there any good kits?
2
votes
0answers
24 views
How to use mechanical microstrainer to extract tissue proteins from human?
Background:
There are many methods to extract proteins form human tissues out there. The majority of them use an extraction buffer containing variable concentrations of detergents and protease ...
1
vote
0answers
17 views
activate a drug using deoxycytidine kinase in vitro
I need to activate a drug similar to the way it is activated in the cell. My set of molecules called Nucleoside analog reverse-transcriptase inhibitors (NARTIs or NRTIs) need three phosphate groups to ...
2
votes
1answer
44 views
Using DTNP to find free thiol groups on a protein
I've been tasked with using DTNB to find the number of thiol groups on a molecule of Bovine Serum albumin (BSA).
After measuring the absorbance, finding the concentration of TNB and calculating the ...
1
vote
2answers
94 views
PEG-silane treatment: why incubate for 18 hours at 60 degrees Celsius?
I am conducting a biochemistry-related experiment and I have been unable to understand a step which is commonly performed.
My aim in this step is to apply a PEG (Polyethylene glycol) silane layer.
...
1
vote
0answers
35 views
Can I heat Trizol?
I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause ...
4
votes
2answers
65 views
Low temperature PCR
We're trying to do emulsion PCR using HA-coated polystyrene beads and we're noticing that the beads are seeing drastic issues with thermal degradation above 90C. As PCR has an unfortunate requirement ...
2
votes
0answers
43 views
Cell growth conditions for preparing electrocompetent cells
Usually the protocol for preparing electrocompetent E. coli cells calls for growing the cells at 37deg and 225rpms until they reach OD of 0.3. I was wondering, is there any reason they should grow at ...
7
votes
3answers
249 views
Why is it sometimes difficult to resuspend E. coli in P1?
It's a curiosity question. When I'm doing minipreps after pelleting the bacteria sometimes it's very easy to resuspend them in P1 (Qiagen kit), but sometimes they form a rubbery clump that is very ...
3
votes
1answer
172 views
Open protocol for Ligase Independent Cloning
Ligase Independent Cloning is a protocol that allow an insert to be integrated into a vector without ligation. It uses T4 DNA polymerase with only ATP to first chew back from blunt ends to create ...
4
votes
2answers
504 views
Why is proteinase K digestion performed at 50 °C?
Many DNA isolation protocols use a Proteinase K digestion to remove proteins. This is often performed at 50 °C. Why is this?
2
votes
0answers
58 views
Has anybody used Evrogen's DSN-normalization protocol for cDNA normalization? [closed]
I found the link to a commercial product by Evrogen to normalise cDNA samples for gene discovery projects here:
http://www.evrogen.com/technologies/normalization.shtml
The most up-to-date reference ...
11
votes
0answers
107 views
How straightforward is in vitro compartmentalization?
in vitro compartmentalization (IVC) is one of those technologies that everyone knows about, talks about, but never actual does due to the rather technical difficulties in setting the system up. I was ...
10
votes
2answers
132 views
protocol for pulldown of DNA breakpoints?
Is there any method to do pulldown enrichment of DNA breakpoints from a cell? I have found this paper reporting a method to enrich for the DNA single-strand breakpoints from meiotic recombination ...
5
votes
2answers
235 views
How to set up a slow cooling on an AB Veriti thermal cycler?
I want to incubate my sample at 50ºC and then slowly cool it down to 4ºC at a rate of 0.1ºC/s. I am using AB Veriti thermal cycler. Does anyone know how to set up the rate of cooling? It has to do ...
