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tantao's Content

There have been 19 items by tantao (Search limited from 30-September 19)


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#113242 problem with poly A adding

Posted by tantao on 22 June 2011 - 02:09 AM in Molecular Biology

Dear everyone,
I use mMESSAGE mMACHINE (AM1344) to in vitro transcription two plasmids ( borh genes are cloned into pcDNA3.1(+) vector). After in vitro transcription (20ul reaction and the plasmid templates are both 1ug) , I use Poly(A) tailing kit to add poly A to the IVT products. Before the adding Poly(A), I take 0.5ul of IVT products(sample1 and sample2) as enzyme minus control, and after adding Poly(A) I take 1ul reaction products form 100ul products, then run non-denaturing-gel. I also quatiation the last products after recovery of RNA by phenol: chloroform ectraction. The results are as follow:
1. before adding the E-PAP Poly A enzyme, I use 0.5ul products from 96ul reaction buffer to run non-denaturing-gel. After adding he adding Poly(A), I use 1ul products to run non-denaturing-gel. However I can not see band in sample 1 before or after adding Poly(A). I can see band before or after
adding Poly(A). The marker is 100bp and the 500bp is 150ng.(figure1)

2. After recovery of RNA by phenol: chloroform ectraction, the RNA is ressolved in 20ul DEPC water I quatiation the last products by UV light absorbance. The concentartion of sample 1 is 5200ug/ml and sample 2 is 5300ug/ml. OD260/280=1.9. However I can see weak band in sample 1, and sample 2 is not like 5300ug/ml, after I take 1 ul to run non-denaturing-gel(figure2).
I do not know what is wrong?

Attached Thumbnails

  • figure 1.JPG
  • figure2.JPG



#90402 problem with MACS cell isolation

Posted by tantao on 25 October 2010 - 02:11 AM in Flow Cytometry

Dear friend,
Increasing the concentration of your antibody appears to best answer, although, the conc. specified by the manufacturer generally works. Try loading not more than 500microl sample onto the column at a time, so that labelled cells can be retaind. Also, I have found that carrying out the steps at 4oC, or in ice, give pretty sepecific results.

I hope this works....I'll dig around a bit and see if I can come up with something better.

Dear maverick3006,
Thanks very much. The concentration primary antibody is 10ug/ml. How do you think about it? too high or too low? This follows is my protocol, would mind giving me some suggestions:
1.Resupend the cells in 200ul of MACS buffer containing primary antibody.
The concentration of primary antibody is 10ug/ml.

2. Mix well and incubate for 15min at room temperature.
When working on ice requires increased incubation times (30min) or 4C for 20min.

3. Wash the cells with MACS buffer for twice (10ml each time). Centrifuge at 300g for 5min.

4. Resuspend the cell in 80ul MACS buffer per 107 cells. Add 20ul microbeads per 107 cells. Mix well and incubate for 15min at 4C.

5.10ml MACS buffer were added and cells were centrifuged for 10 minutes at 300 g. Discard the supernatant and resuspend the cell with 500ul buffer.

6. Rinse the the column (MS) with 500ul MACS buffer. Apply the cell suspension into the column.Wahsing the column for three times (500ul/each time for MS column)
Only add new buffer when column reservoir is empty.

10. Add the 1ml MACS buffer into the column reservoir, flush the cells by pushing the plunger into the column.

11. Centrifuge the cells at 300 g. for 5min, resuspend the cells in 1ml culture medium. Plate the cells into culture plate.



#90388 problem with MACS cell isolation

Posted by tantao on 24 October 2010 - 11:11 PM in Flow Cytometry

Dear everyone,
we use MACS to isolate the positive cells.But after isolation the positive cells are all in the depletion part. I do not know why, and someone told me if the concentartion of primary antibody is too low or high. The MACS does not work! IS that true? Shall I titre the antibody? Thanks very much!



#90386 problem with MACS cell isolation

Posted by tantao on 24 October 2010 - 11:10 PM in Immunology

Dear everyone,
we use MACS to isolate the positive cells.But after isolation the positive cells are all in the depletion part. I do not know why, and someone told me if the concentartion of primary antibody is too low or high. The MACS does not work! IS that true? Shall I titre the antibody? Thanks very much!



#85184 PCR contamination

Posted by tantao on 30 August 2010 - 06:02 AM in PCR, RT-PCR and Real-Time PCR

55C is not too high, although not too low either. If possible try a gradinent PCR, or if you cannot, try 60 C with a real sample and the contaminated water....

edit: try to clean the pipett in the inside as well!

Dear gebirgsziege,
how to clean the pipett in the inside. I use 75% alcohol to clean the pipett. Is it ok? Thanks again!



#85178 PCR contamination

Posted by tantao on 30 August 2010 - 05:54 AM in PCR, RT-PCR and Real-Time PCR

Your primer stock could be contaminated. For PCR primers, I usually keep a concentrated stock and a more dilute working stock. Do you have a more concentrated stock that you can make a new working stock from? This would be a quick test

Dear Kaioshin,
I order the new primer and use the new one to run the PCR. but the ghost band is still there.



#85177 PCR contamination

Posted by tantao on 30 August 2010 - 05:52 AM in PCR, RT-PCR and Real-Time PCR

sorry so my post was then not much help for your problem, sorry for this.

It looks as if you got a contamination in your chemicals, have you tried to change them and are you sure that nobody else used your chemicals?
And second: autoclaving does not necessary destroy DNA contamination on you pipetts etc., you should try and clean them with bleach, DNA-off or something similar. Do you have some of purchased ultraclean certified DNA/RNA free water in your lab (often also comes as component of extraction purification kits)? Try to use this water for the negative control, if possible try to do your PCR with chemicals from another lab that does not work with mice using their equipment....

Last but most important:can you post your PCR protocol? If the annealing temp is too low you might get unspecific priming with some DNA contaminiation. If possible run a gradient PCR with water only to see at which temp the band is disappearing. If the times are too long there might also be a problem with specificity.

gebirgsziege,Thank you very much! Only me use these buffers. and I also use UV to bleach the pipetts for 30min
my PCR protocol as follow
1.95℃ 2min
2.95℃ 30s
3.55℃ 30s
4.72℃ 30s
repeat step 2-4 35 times
5.72℃ 10min



#85167 PCR contamination

Posted by tantao on 30 August 2010 - 05:37 AM in PCR, RT-PCR and Real-Time PCR

proceed with your experiment sometimes it will work, when I got the same problem i went forward it was fine

Dear ranvi,
I used this primer for real-time pcr. There is not dimer and false positive before. But now everything comes out.



#85154 PCR contamination

Posted by tantao on 30 August 2010 - 05:01 AM in PCR, RT-PCR and Real-Time PCR

when trying to amplify DNA from Bacteria, contamination from Taq production (=DNA from ecoli) in the enzyme preparation can be the problem.
We once had exactly this problem, but after a lot of asking and trying using either Phusion (Finnzymes) or TrueStart (Fermentas) instead of the routinely used cheap enzyme solved the problem.

Dear gebirgsziege and everyone,
I repeat the PCR again. I amplify the beta actin of mouse. I use the ddH2O as template. But I also get a band around 100bp which is the correct band. I am really crazy. What is wrong!!!

Attached Thumbnails

  • 1.JPG



#85137 PCR contamination

Posted by tantao on 30 August 2010 - 02:06 AM in PCR, RT-PCR and Real-Time PCR


I saw someone mentioning gloves which resulted in contamination in this forum. Use separate pair for gloves to prepare your mastermix and to aliquot it into tubes. Also, get a brand new set of primers (it might be contaminated).

Ameya


If you prepare your master mix in a flow cabinet, make sure that it is completely clean and maintain the filter off.

CRIS

Thank you Ameya and CRIS. I always make the flow cabinet open. Maybe this the problem. I run the PCR again. Hope everything is ok this time.



#85102 PCR contamination

Posted by tantao on 29 August 2010 - 08:12 PM in PCR, RT-PCR and Real-Time PCR


Dear everyone,
The PCR contamination makes me crazy. My band is 107bp, I use ddH2O as template and get the positive result. I change everything: the PCR buffer ,ddH2O and use the filter tips. I also autoclav the pipette. But when I use ddH2O as template ,the band are still there. I do not know what to do know. Can anyone give me some suggestions?
Yours sincerely
Tao



For starters, you could try ordering primers that are slightly different than the ones you have. Shifted a couple, 5, or 10 bp from the ones you have now. You could be dealing with some type of primer dimer product rather than a contamination of template.


Thank you Kaioshin. the primer dimer is below 100bp. I do not know what is the problem.



#85089 PCR contamination

Posted by tantao on 29 August 2010 - 07:07 PM in PCR, RT-PCR and Real-Time PCR

Dear everyone,
The PCR contamination makes me crazy. My band is 107bp, I use ddH2O as template and get the positive result. I change everything: the PCR buffer ,ddH2O and use the filter tips. I also autoclav the pipette. But when I use ddH2O as template ,the band are still there. I do not know what to do know. Can anyone give me some suggestions?
Yours sincerely
Tao



#81289 help! there is insoluble matter in PCR product

Posted by tantao on 27 July 2010 - 03:53 AM in PCR, RT-PCR and Real-Time PCR

Dear everyone,
I am doing single cell PCR these days. However after PCR, there is white insoluble matter in PCR product, I have never seen before. I do not know what is the problem. Could you help me. Thanks very much! I am crazy.



#80179 problem about pIRES2-DsRed2 double digestion

Posted by tantao on 19 July 2010 - 08:07 PM in Molecular Cloning

This is probably some minor star activity of one of your enzymes. Don't worry about it. It looks as if your ligations worked. Forward. If it worries you, turn down the exposure time on your gel images.


Dear phage434,
Thanks very much! the enzymes in my double digest buffer have no star activity.However the activity of XhoI in this buffer is 50-100.I really do not know what happened!
sincerely
Tao



#80027 problem about pIRES2-DsRed2 double digestion

Posted by tantao on 19 July 2010 - 12:23 AM in Molecular Cloning

Dear everyone,
I have a problem about pIRES2-DsRed2 (5.3kb,clotech) double digestion. I used XhoI and BamHI to digest the pIRES2-DsRed2 and I got two bands, which are both around 2500bp. I should to be only one band. And then I extracted the fragment (5.3kb) from the gel. I ligated this fragment with my insert fragment(700bp, digested with same enzymes from another plasmid). I transformed the bacteria and picked up the colony. I redigested the plasmid again with same enzymes. To my suprise, these two bands (around) appeared again. After that I use same plasmid( pIRES2-DsRed2 )from another lab , I got same phenomenon. I do not know what happened! I am crazy , could you give me some suggestions.


figure 1 lane 1 is the marker ; lane 2 is pIRES2-DsRed2 double digestion with XhoI and BamHI ; lane 3 is another plamid double digestion with XhoI and BamHI . arrow head indicate these bands around 2500bp.

figure2 lane 1 is marker. lane 2-6 is ligated plasmid double digestion with XhoI and BamHI . All of them have my insert fragment.arrow head indicate these bands around 2500bp.

Attached Thumbnails

  • figure1.JPG
  • figure_2.JPG



#79432 problem about vector Dephosphorylation

Posted by tantao on 14 July 2010 - 01:13 AM in Molecular Cloning

on the NEB website for T4 DNA ligase it says this in part of the description:

"...This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1)."



Thank you very much molstudent!



#79387 problem about vector Dephosphorylation

Posted by tantao on 13 July 2010 - 08:19 PM in Molecular Cloning

Dear everyone,
I have questions about the vector dephosphorylation and ligation. When I digest the vector with enzyme, in order to avoid the vector self-ligation, the vector was dephosphorylate. Then I digest the insert fragment from another vector and ligate them. But I think maybe there is problem, the insert and the vector only form two phosphodiester bonds and the other two sites are nicks, they can not form phosphodiester bond. if when this product transfects into the E.coli, the bacteria will repair these two nick? Could you give me some suggestions, Thanks in advance.



#77581 problem about DNA electrophoresis

Posted by tantao on 30 June 2010 - 12:21 AM in Electrophoresis

Thanks very much, fysio lab ,home brew and vladooo. The phenomenon is that I see two bands with an unstained middle part when look at the gel slice in vertical cross-section. So I think I should strain the gel more longer. Thanks everyone again !



#77296 problem about DNA electrophoresis

Posted by tantao on 28 June 2010 - 01:45 AM in Electrophoresis

Dear everyone, I have a problem about the DNA electrophoresis. when I use the big well to run the DNA electrophoresis(in order to extraction the pcr product from gel), the sample can not settle down completely. The DNAs become two layers and there are no DNAs in the middle of the gel. When I use the small well, everything is ok. I do not know what happened, could you give me some suggestions! Thanks very much!




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