Problem with expanding Gecko library--recombination problem.

[Wenhua Gao]

Did anyone have the same problem as what we saw here? We saw two bands after maxi prep and it seemed that the lower one is recombined. Thanks a lot for any suggestion. Here is what we did:

We electroplated Lucigen E. cloni 10G Elite cells according to the company’s protocol.   4X 100ng transformations in 25ul bacteria each time.  1 mL SOC was added immediately following transformation, and then added to an additional 1mL SOC at room temperature and recovered at 37 for 1 hour.  We then pooled the 8mLs of bacteria and distributed it on 15, 145mm dishes with LB amp agar.  We let the plates dry, and let them grow overnight, and then scraped as described in the protocol.  We spun down the pellet and it weighted in at 3.6 grams. We used triple the recommended Qiagen solution, and obtained 2mg of DNA.  Here is the gel:

[Neville Sanjana]

Hi Wenhua,

We normally get a single (large) band after digestion with a single-cutter (ie. to linearize plasmid). Can you try linearizing the plasmid? Sometimes if you run uncut DNA after plasmid prep, you can see multiple bands due to different DNA forms but cutting should make it all linear.

Also, did you put all of the bacterial pellet (3.6g ) on a single maxi column? I think Qiagen recommends much less (<0.5g) per maxi column.

- Neville

On Sat, Mar 22, 2014 at 11:52 PM, Wenhua Gao <gao...@gmail.com> wrote:

Did anyone have the same problem as what we saw here? We saw two bands after maxi prep and it seemed that the lower one is recombined. Thanks a lot for any suggestion. Here is what we did:

We electroplated Lucigen E. cloni 10G Elite cells according to the company’s protocol.   4X 100ng transformations in 25ul bacteria each time.  1 mL SOC was added immediately following transformation, and then added to an additional 1mL SOC at room temperature and recovered at 37 for 1 hour.  We then pooled the 8mLs of bacteria and distributed it on 15, 145mm dishes with LB amp agar.  We let the plates dry, and let them grow overnight, and then scraped as described in the protocol.  We spun down the pellet and it weighted in at 3.6 grams. We used triple the recommended Qiagen solution, and obtained 2mg of DNA.  Here is the gel:

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[Jason Yuan]

 Hi Wenhua, have you solved the problem? We met exact the same one here...

Jason

[VajraDog]

I'm having the same problem too. I did use BamHI digest but still ended up with 2 bands, just like Wenhua.

-Vajra

[Moli Joshi]

We are having the same problem...Any ideas/suggestions on how to fix this?

[Neville Sanjana]

Dear Jason, Vajra, and Moli:

I have a few thoughts…. It looks like the upper band (presumably the one without any recombination?) is much stronger in the maxi prep (based on the gel images I have seen from a few of you). I still am not sure why there is a lower band, as we do not see it in our preps, but I think it’s probably fine to make virus. After you make virus, I can think of two possibilities:

1) Plasmid from the lower band is missing vital viral elements such as RRE, the gag hairpin after the 5’ LTR, cPPT, etc. and thus virus is only produced from the upper band plasmids.  Net result is these plasmids will not affect your GeCKO screen.

2) Plasmid from the lower band results in virus but, given your sequencing results, the virus does not confer Puro resistance. No cells with this virus will survive puro and hence these plasmids will not affect your GeCKO screen.

Before making virus, I think it is probably a good idea to PCR amplify the sgRNA cassette from the upper band (similar to the procedure for reading out the library after screening, see our Supp Methods) and deep sequence it. This way you can verify that you have cloned with adequate representation to keep most of the sgRNAs. 

Good luck!

- Neville

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[Neville Sanjana]

Another GeCKO user just wrote to me with data that shows that library retransformation with a lower growing temperature can help reduce recombination. Please see his figure after single-cutter enzyme digest of maxiprep DNA. The lower band is an undesired recombination. Also note his use of more recombination-resistant Endura electrocompetent cells from Lucigen. I have also been using these cells and they work great.

Hope this helps some of you who are having issues with lentiviral recombination.

- Neville

[Molishree Joshi]

Hi,

We tested the Lucigen Endura, but are still seeing significant recombination. 

There is no doubt that the Endura cells are significantly better than E.cloni cells and setting overnight cultures 30C for 15hrs looks better than 37C for 15hrs. However, there is significant recombination.

Any ideas why and how we can address this?

Thank you.

-Moli

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-- Molishree Joshi

355 27th St,

Boulder, CO 80305

[Neville Sanjana]

Hi Moli,

Sorry to hear that you're still having troubles. It does seem like you're seeing more recombination that what we see (which is very very little). Since recombination likely destroys the Puro cassette, I think you will be fine (see my reasoning further up in this thread). You could also gel purify this recombined band and verify by sequencing that Puro is no longer present. 

Good luck!

- Neville

[saul kivimae]

Invitrogen has stbl2 and stbl3 competents worth trying. I don't know how different they are from lucigen cells. Compare the annotated genotypes.

I do lenti propagation in these cells with vectors that completely fall apart in "regular" competent cells.

Saul

[elif karaca]

Hi,

I have expanded Gecko library v2 as in protocols and also used Endura cells at 32 degree for 14hrs. But when I digested my library, I saw different patterns with EcoRI and BamHI digestion ( even though both of them single cutter). If I consider only BamHI, then there is no recombination but if I consider EcoRI, there is extra bands. I also did double digestion. So now I do not whether I should start virus production, or try to expand again with 30 degree.

Did anybody try to produce viruses even though there is recombination? I know Neville said it will not affect virus production but I wanted to double check.

Many thanks,

Elif

[Neville Sanjana]

Hey Elif,

Your single digests look good to me. The extra bands in the EcoRI digest look much lighter than your main plasmid band.... perhaps there is a little star activity with this enzyme? As you say, the BamHI digest looks perfect. I do recommend sequencing your plasmid to make sure representation (library complexity) looks good.

- Neville

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