7 replies to this topic

[mickey]

Dear u guys,

I recently made a three pieces ligation into a mammalian expression vector I usually used. After transformation I pick up colonies to do miniprep for digestion diagnosis and sequence.

But when I use miniprep kit (GE) like before according to the manual, I got very very smear and faint band although the size seems as predicted. I got enough Ecoli cultured for 16 hrs in 5 ml LB to undertake the Alkaline Lysis step,  And throughout the process of miniprep, there is nothing weired about pellet thing.

After I got the bad miniprep construct, I run PCR to comfirm the insert, and it seems OK. But when I pick up the colony again, it gave same smear and faint band.

Anyone else met this occation before? There is some specific targeted degrading system in the E coli to make such a phenomen? Becaue I do this experiments kind of lot, but this smear thing never came up.

[Curtis]

Did you add RNase at all? in the first solution or the elution buffer?

[perneseblue]

could we have a picture of the gel?

Was a sample of the cut with restriction enzymes to see if it gave correct banding patterns?

[mickey]

perneseblue, on 29 October 2010 - 08:02 PM, said:

could we have a picture of the gel?

Was a sample of the cut with restriction enzymes to see if it gave correct banding patterns?

Yeah the pattern seems right. but too much smear

Attached Thumbnails

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[mickey]

Curtis, on 29 October 2010 - 07:42 AM, said:

Did you add RNase at all? in the first solution or the elution buffer?

RNase is contained in the resuspending buffer of the kit.

[phage434]

I think the major problem is a bad gel, rather than an incorrect product.  You should use a lower percentage gel to get better separation of fragments of the size you care about, 0.5% to 1%.  For 5000 bp fragments, you probably want an 0.8% gel.  Also, you appear to be adding too much stain to the gel, resulting in high background, and adding no dye to the positive terminal buffer.  Since the dye runs to the negative terminal, this leaves a portion of your gel unstained (bottom part), while the top part is highly stained (top).  I would do one of two things, either stain after running the gel (probably best, but more tedious), or fix both problems with your current strategy, by adding less dye to your gel, and also adding a similar amount of dye to the positive buffer pool in your gel running apparatus.

[perneseblue]

could you tell us about the strain of E coli used to grow this plasmid?

[123blockhead]

mickey, on 28 October 2010 - 10:18 PM, said:

Dear u guys,

I recently made a three pieces ligation into a mammalian expression vector I usually used. After transformation I pick up colonies to do miniprep for digestion diagnosis and sequence.

But when I use miniprep kit (GE) like before according to the manual, I got very very smear and faint band although the size seems as predicted. I got enough Ecoli cultured for 16 hrs in 5 ml LB to undertake the Alkaline Lysis step,  And throughout the process of miniprep, there is nothing weired about pellet thing.

After I got the bad miniprep construct, I run PCR to comfirm the insert, and it seems OK. But when I pick up the colony again, it gave same smear and faint band.

Anyone else met this occation before? There is some specific targeted degrading system in the E coli to make such a phenomen? Becaue I do this experiments kind of lot, but this smear thing never came up.

have you heard of Dnd (DNA degradation) phenotype? as i know, Dnd phenotype are found in many strains of bacteria, including E. coli