TRUE postives or non specific bands, pls help

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: Leonid, amiradm, BioTeam

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

TRUE postives or non specific bands, pls help

Post by kandyan » Fri Jan 08, 2010 12:42 am

Please let me know if any of the lanes in the gel picture contains a positive. the band size is 300 bp. I do see a faint band in right place in lanes 5 6 7 8 11, but there are non specific bands too,,,, pls help
SF 4 1 10.JPG
P1040114.jpg
Last edited by kandyan on Fri Jan 08, 2010 10:28 am, edited 1 time in total.

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Re: TRUE postives or non specific bands, pls help

Post by JackBean » Fri Jan 08, 2010 7:48 am

At least you could write up, what marker you have :roll: :x I guess, the most visible band is 500 and the second one is 100?

Even your marker does not look very well ;) You may try to change your polymerase to some more robust for your application. Do you have some positive control?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

Post by kandyan » Fri Jan 08, 2010 9:36 am

Thank you!
The marker is in lane 16 which has not worked well!!! Only the 500 bp one is visible. Positive sontrol is in lane 1. It has worked nicely. I want to know about lanes ,4 5 and 7. They have multiple bands but there is a band at 300 bp or is is my imagination?

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Fri Jan 08, 2010 10:06 am

Yeah, I recognized the marker, but my point was, what kind of marker it was. I guess some 100bp from Invitrogen?

Don't you think, that the band in line 1 is smaller, than in other lines? Maybe the one in line 12 could have the same size... But I don't think it's 300bp...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

Post by kandyan » Fri Jan 08, 2010 10:29 am

Sorry aboyt the mis communication, yes it is an invitrogen 100 bp marker. I re ran the gel after dilution, now the suspected lanes are next to the ladder, (2nd phot uploaded) what about it please?

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Fri Jan 08, 2010 10:38 am

OK, now I see some bands of the same size as in the first line (but I don't recognize the marker, sorry).

But if I were you, I would care about the non-specific bands. What is this amplification? What are you doing?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

Post by kandyan » Fri Jan 08, 2010 11:40 pm

The marker is in the 4th lane. I trying to establish a diagnostic PCR for an intracellular bacteria.
The assay was validated with the positive control. But, the samples give poor results. I think it is something to do with the DNA extraction. By any chance do you know about a good manual extraction method for intracellular organisms when using whole blood?

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Sat Jan 09, 2010 10:05 am

Did you check, whether are the primers complementary to human genome?

Try other polymerases, even your positive control does not seem very well. You should also include some negative control (water, but more importantly some sample, where you for 200% don't have your bacteria;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

Post by kandyan » Sat Jan 09, 2010 11:27 am

Thank you, will try a different Taq and see. No, there is no complementarity to human gemone. Negative controls are always done, lane 2 in both pics. Will do with a different Taq and see. Thanks again!

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Sat Jan 09, 2010 11:29 am

What do you use as negative control? Water?

What is the GC percentage of your sample? How many step cycling are you using?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

kandyan
Garter
Garter
Posts: 7
Joined: Fri Jan 08, 2010 12:32 am

Post by kandyan » Sat Jan 09, 2010 1:33 pm

Negative control is ultra oure water
Cycling numbers 35
GC percentage is within accepted, the thing is this assy has worked very well previously!!!!

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Sat Jan 09, 2010 1:41 pm

Use negative control from non-infected human.

Well, than you should check out, what has changed between tests, where it was fine and where it was smeared ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Post Reply

Who is online

Users browsing this forum: No registered users and 2 guests