Dear all,
I want to isolate DNA from mouse lungs to check for gene deletion (The Cre is expressed specifically in Type II alveolar cells).
I tried to isolate the DNA using the method discussed below but could not see any band after PCR.
No bands are seen for flox and Cre as well from the lysates.
Can someone pls share their protocol or any suggestions.
I tried the following:
Extracted lung from 9 week old mice, right lobes were minced in Lysis buffer (50mM Tris (pH 8), 100mM Nacl, 0.1% SDS, 500ug/ml proteinase K and 20ug/ml RNAse) and were incubated at 55C for overnight (roughly 16 hrs).
The lysate was viscous and appeared brown color. I heated the sample to 92C to inactivate proteinase K and precipitated the DNA with ice-cold isopropanol.
I obtained a very big gluey pellet which I washed with 70% ethanol, dried, resupeneded in water. The pellet didn't dissolve. The A260 was 300ng/ul (A260/280= 1.87).
The PCR did not work !
I would really appreciate your responses.
Many Thanks
Apoorva
hi. Dr. Apoorva Verma
i suggest you these:-
1- Genomic DNA Preparation from RNAlater™ Preserved Tissues
https://www.thermofisher.com/in/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/genomic-dna-preparation-from-rnalater-preserved-tissues.html
2-Genomic DNA extraction from tissues animal
https://www.kurabo.co.jp/bio/biodocument/eng/QuickGene_Application_Data_DA-b_Genomic%20DNA%20Extraction%20from%20Tissue%20of%20Animal.pdf
3- A simple, rapid method for isolation of high quality genomic DNA from animal tissues
https://academic.oup.com/nar/article-abstract/23/24/5087/2400744?redirectedFrom=PDF
good luck
Agreed with all above. However there are too many moving parts.
Fistly, did your positive and negative controls in PCR work?
Hello Apoorva. I have used a quick and dirty method to isolate genomic DNA from tail, kidney, and spleen for genotyping, and I believe this should also work for your purposes.
To each 1-3 mm tail biopsy (in this case, use a small biopsy of the lung), add 100 uL of lysis buffer (see below), 2.5 uL of proteinase K, 5 uL of Igepal, and 5 uL tween 20. Incubate at 55C O/N, and then 20 minutes at 95C (I use a PCR machine for these steps). Afterwards, use 1-3 uL of the supernatant in your PCR reactions.
***It is important to note that too much tissue can be inhibitory to the PCR reactions, so make sure to not add too much.
Good luck!
Lysis buffer:
50 mM Tris pH 8.0
50 mM KCl
2.5 mM EDTA
Add fresh:
Tween 20 to 0.5% 10% stock
Igepal (NP40) to 0.5% 10% stock
Proteinase K to 250-500 ug/mL 20 mg/mL stock
Hi Thanks a lot for your helpful suggestions- Avinash Marwal , Ahmed Housanie Elsappah & Ozkan Gelincik
Rithy Meas Thanks. I will try the protocol and will update you the results. Hopefully it works :)
Hey Apoorva Verma , just checking back in on your issue. Did my protocol end up working?
Hi Rithy,
Many Thanks for your suggestions. I routinely perform DNA isolation from mouse tails and get good PCR products for Cre and Loxp & other genes. I tried your protocol on small pieces of lungs but it gave me different bands for LoxP, Cre and YFP as opposed to tail samples. For example, Tail sample gave me two bright bands for YFP gene (hetero) and lung sample gave me just 1 faint band. I am not very sure if it is working correctly.
- Badges
- Science method