shawn6022

Hey again everyone,
I have a feeling I am going to be posting a lot on here. This website has become a very valuable resource for me lately.

Well, the recent snag is that I am trying to do crossover PCR to delete a gene. I have both the 3' and 5' pieces. I amplified the 5' piece with ease..first try. The 3', however, was a bit complicated...the Tm for the SOEing forward and Rev was calculated to be 68 C but I ended up having to increase the actual temp in the thermocycler to 72 C due to non-specific binding. Well, I got the 3' eventually and now I am at the 2nd PCR where I am adding the two pieces together. This is where it gets odd. The 5' piece is 1.2 kb and the 3' is 1.3 kb and after I run the PCR, and analyze on the agarose I keep getting a huge band at 1.2-1.3 kb...I get a very faint band around 2.5 kb..which is where the band should be. The annealing temp for the for and rev is 68 C so I have ran the PCR at increasing temps from 63-67 C and as I increase the temp the faint band at 2.5 kb gets fainter and fainter as the temp increases.

I have uploaded a picture.

Lane 1 is Molecular weight marker- 2 log ladder from NEB
Lane 2 and 3 are the SOEing PCR after the second round. I do everything in doubles in case I screw one up...they both were the same conditions...

I run 25 uL reactions initially until I get the kinks worked out. For the master mix I add:

18 uL ddH2O
0.125 uL taq
0.5 uL forward primer
0.5 uL reverse primer
1 uL DMSO--I work with Pseudomonas which is GC rich
0.5 uL dNTP
2.5 uL Taq Buffer

and then I add 1 uL of each piece (5' and 3' after gel extraction and running on an agarose gel to make sure I didn't lose it)...

Do you have any suggestions as to what I can do to get the correct amplification?
 UVP01173Apr122013.jpg   32.69K   17 downloads

Hey everyone,

I am trying to extract a plasmid from E. coli that has my vector and SOEing insert already ligated in. I am using the alkaline lysis method and here lately I have been extracting nothing but genomic DNA (DNA at the top of the gel near the well) and not plasmid..I am running the plasmid on an agarose gel before restriction digest. I am just recently having problems with this. I isolated a plasmid last November using the same method with little to no problems.

Here is the basic protocol I am using:
1. Grow culture overnight in 5 mL of LB with antibiotic (in this case its ampicillin)
2. centrifuge 1.5 mL for 5 minutes and remove all media
3. Resuspend pellet in 150 uL TE and let sit for 5 minutes
4. Add 300 uL if fresh 10 M NaOH and 10% SDS and 3 uL of RNase, mix by inverting 6 times, and let sit for 3 min
5. Add 225 uL Pottasium acetate, and mix by inverting 6 times
6. Centrifuge for 10 minutes
7. Transfer supernatant to new tube and extract with an equal volume of choroform, phenol, isoamyl alcohol
8. Vortex and centrifuge for 5 minutes
9. Transfer supernatant to a new tube
10. Add 2 volumes of cold 95% EtOH and leave on ice for 25 minutes
11.Centrifuge for 5 minutes
12. Remove supernatan and drain tubes on a paper towel by propping them up on a sharpie to allow the EtOH to drain off
13. add 1 mL cold 80% EtOH, and centrifuge for 5 minutes
14. Remove supernatant, drain well nad let dry for 10 minutes
15. Resuspend in 50 uL of TE..

That's what I did last November also when I was not having any problems...and now I am isolating genomic DNA. Any suggestions?

hello everyone,

I am having trouble going to KEGG from the NCBI website... when I look up my gene of interest on NCBI I try to follow the links to KEGG to get the sequence but every time I try it sends me to an error screen saying that the page can't be loaded..has any one else had this problem? Are there any other websites that I can go to get the sequence? Thanks for your help..

Shawn

Hey everyone,
I am a fairly new grad student so I'm still learning a ton of tricks and getting a plethora of new projects to work on. My latest involves screening mutants that were created by a former grad student. This library was created using the mariner transposon. My P.I. suggested that I do rescue cloning to try and identify the genes that were disrupted. I tried looking up how to do this online and haven't really come across any protocols on how to do it. He gave me the basics but I would feel more comfortable if I got more specific details about it. Can anybody help out?
Thanks!

Hello everyone!
I just started grad school the beginning of this past semester, and I am relatively new to the site also. Anyway, I just started a cloning project and my p.i. told me to use a specific plasmid to use. The issue I am having is that I cannot find a map of the plasmid pJDC15. I have the papers where its construction is described but I have no idea where to actually find a restriction map. Does anybody have any kind of leads or know where I can find it?
Also, I was wondering if anybody had any sage wisdom they would like pass on to me in regards on how to survive grad school..

Thanks in advance and I hope you have good day..