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Janina's Content

There have been 26 items by Janina (Search limited from 11-November 18)



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#9393 Stability of DNA at 4 degrees?

Posted by Janina on 14 December 2004 - 02:05 AM in Molecular Biology

In general, DNA can be stored at 4 C, either in buffer or water... But this depends on your DNA. E.g., some viral DNA is not stable in the fridge.



#9269 How to insert a Tag to the middle of a protein?

Posted by Janina on 08 December 2004 - 09:03 AM in Molecular Biology

It is a very complex thing to explain... Well, PCR allows you to insert fragments into your DNA. Therefore you need primers which contain a part of your DNA, a part where a restriction site (rs) is encoded and some overhangs (to be sure that your rs works). If your DNA allready contains a rs, you only need to amplify your fragment with this rs. But caution: it is better that every end of you fragment has its own rs! Otherwise, it is possible that your fragment may recirculate and you are not able to ligate it... Also, your DNA must have - of course - same rs as your fragment...

Hope this will help a little bit... :D



#9216 ligation and transformation

Posted by Janina on 06 December 2004 - 11:55 PM in Molecular Biology

I think, any antibiotic is not stable for a long period of time at 4C or even RT... Be sure to use a fresh aliquot. I am careful so that I change aliquots every 2 - 3 weeks in the fridge.



#9215 ligation and transformation problems! Urgent!

Posted by Janina on 06 December 2004 - 11:49 PM in Molecular Biology

And what did you change at least?



#9184 ligation and transformation problems! Urgent!

Posted by Janina on 06 December 2004 - 12:24 AM in Molecular Biology

I don't think that it is necessary to purify by gel after first digestion. The loss of DNA could be to high... But it is necessary to refresh buffer after first digestion.



#9128 ligation and transformation problems! Urgent!

Posted by Janina on 03 December 2004 - 01:49 AM in Molecular Biology

I do a digest at 37 C for 3 h, 10 min aT 65 C... Perhaps it is better to do a sequential digestion.
But I make no difference in incubation time between vector and insert.



#9099 BamH1 and Nde1 double digestion problem

Posted by Janina on 02 December 2004 - 08:22 AM in Molecular Biology

Try following link:
http://www.fermentas...gest/index.html

It is from MBI Fermentas but you can also see how fine they work together. Perhaps you can also compare buffer condition.



#9098 ligation and transformation problems! Urgent!

Posted by Janina on 02 December 2004 - 08:18 AM in Molecular Biology

Although protocol tells 5 min at RT, I do ligate with such fast kits up to 15 min... It is also necessary to mix/vortex reaction (and spin down) before incubating.

Perhaps in your calculation something is wrong... Did you measure conc by photometer?



#9087 ligation and transformation problems! Urgent!

Posted by Janina on 02 December 2004 - 02:27 AM in Molecular Biology

I have 100 ul competent cells, fill them during the protocol with 400 ul LB and plate 50 and 150 ul.



#9086 A Digestion problem

Posted by Janina on 02 December 2004 - 02:22 AM in Molecular Biology

This is right, the extinction of DNA (260 nm) should be between 0.1 and 0.01 - otherwise the result won't be precise enough.

For ligation, I use fmol (short calculation from ng to fmol...). Normally 5 fmol vector, 15 fmol insert. It is always the best to use as less ng as possible because the competent cells don't like so many DNA and the efficiency might be less... :rolleyes:



#9038 ligation and transformation problems! Urgent!

Posted by Janina on 01 December 2004 - 12:59 AM in Molecular Biology

Hace you tested your cells (efficiency, positive control)? Have you plated a negative control (religation)? Have you used new LB-Kan-plates? Someone in this forum wrote that Kan is reduced faster than "normal" antibiotics...

I use only 2 ul for transformation, too. The more is often not the best... especially in mobi... :unsure:



#9037 A Digestion problem

Posted by Janina on 01 December 2004 - 12:45 AM in Molecular Biology

It may be better to elute DNA with water, so that enzymes have the right buffer concentration - some enzymes are very sensitive to the conditions...

I think, it is always better to calculate the right ratio. I would always measure DNA-concentrations for ligation!



#9036 Small competent cells

Posted by Janina on 01 December 2004 - 12:37 AM in Molecular Biology

I compared efficiency of "old" and "fesh" competent cells, both for alpha and nova. And both fresh cells grow slower... This method seems to work, but the cells have to grow more than 16 hrs to result in a good size for picking.

Well, I'll try Maniatis method next time, I guess. :unsure:



#9007 Where are you from?

Posted by Janina on 30 November 2004 - 04:17 AM in Chit Chat

Cologne, Germany



#9006 Small competent cells

Posted by Janina on 30 November 2004 - 03:47 AM in Molecular Biology

The competent E. coli cells I produced grow very slow. I tested DH5 alpha and Nova Blue, both grow slower than before.
I grow an overnight culture in 2 ml LB, then inocculate 1 ml in 100 ml LB + 10 mM KCl + 20 mM MgSO4 and grow them to an OD600 = 0.5. After this, I cool them down on ice for 10 min, centrifugate 5 min at 2800 g, 4 C, resuspend pellet in 40 ml buffer I ( 30 mM Potassiumacectate, 50 mM MnCl2, 10 mM CaCl2, 100 mM RbCl, pH 5.8) and incubate again on ice for 10 min. Next steps are: centrifugating 5 min, 2800 g 4 C, resuspending in 4 ml buffer II (10 mM MOPS, 75 mM CaCl2, 10 mM RbCl, 15 % v/v glycerol, pH 6.5) incubating 10 min on ice, freezing in pre-cooled tubes.

Now I don't understand why the stains, treated by same procedure, result in smaller colonies.
Any suggestions? :unsure:

Thanks



#9003 Low copy number of plasmid

Posted by Janina on 30 November 2004 - 12:58 AM in Molecular Biology

Try 10 ml culture! It works most of the time fine... Then elute with warm buffer/water (I use 30 C) - this will increase the yield.



#8919 His-tag protein purification - contamination with other...

Posted by Janina on 26 November 2004 - 12:20 AM in Protein and Proteomics

Me too.



#8918 Pfu

Posted by Janina on 26 November 2004 - 12:16 AM in Molecular Biology

Did you check the buffer? It has to be with MgSO4...



#8683 Restriction enzymes cut reaction

Posted by Janina on 17 November 2004 - 02:05 AM in Molecular Biology

I have also problems with the pET22b vector... We found out that it is possible to cut by sequentiell digestion but not by double digest. Perhaps this will help you...



#8650 ligation of pcr product

Posted by Janina on 16 November 2004 - 02:26 AM in Molecular Biology

In my case, it is a restriction site. It is BalI/MscI/MlsI ... But now, we seem to be close to the solution of our problem... Some little experiments left to do... :rolleyes:



#8623 ligation of pcr product

Posted by Janina on 15 November 2004 - 12:22 AM in Molecular Biology

Hi Arwen!

Did your blunt end ligation work? I am asking because I have several problems with my cloning... My Insert seems to be ligated (controlled by Colony-PCR) but then my blunt end site does not work anymore... So perhaps, it is a general problem with blunt end ligation... :unsure:



#8425 His-tag protein purification - contamination with other...

Posted by Janina on 04 November 2004 - 06:13 AM in Protein and Proteomics

You could also put some Imidazole in your Binding Buffer so that these other His-proteins may not bind that much... I used a concentration of Imidazole of 5 mM in Binding Buffer, 60 mM in Wash Buffer and 1 M in Elute Buffer.



#8422 losing protein activity

Posted by Janina on 04 November 2004 - 04:36 AM in Protein and Proteomics

What about degradation of your protein by your bacteria? Perhaps they don't like it... Or check the protein folding resulting of the sequence (degenerate codons?)...



#8417 frameshift after ligation

Posted by Janina on 04 November 2004 - 02:56 AM in Molecular Biology

Thanks for responding that quick.

But the point is that we have one base pair too much, so that the possibility of a exonuclease contaminant doesn't fit.



#8375 frameshift after ligation

Posted by Janina on 03 November 2004 - 09:10 AM in Molecular Biology

We tried to clone a 700 bp fragment, amplified with PCR, in our 5.4 kb vector. One main problem is, that both are digested with EcoRI and MscI (blunt end). So therefore, we dephosphorylated the vector by using CIAP, we ligated the insert in a 1 : 3 molar ratio with some more units of ligase, with PEG and also overnight at 16 C and transformed it in E. coli Nova Blue. We picked some clones for doing a colony PCR and then we let the positive ones grow over night.
Well, now the big main problem is, that we are not able, to cut out the insert of our positive clones, which have the insert for sure...! The blunt end restriction site MscI is no longer working. And so I am asking myself, is it possible that the enzyme MscI might cut a kind of sticky end with 1 bp overhang (however), so that the insert might be ligated with a single-strand break and the bacteria fill the missing base?!? Might the sequence TGGCCA be susceptible to mutations?
I would be glad for further information... :unsure:




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