Low yield for lentiSAMv2 and lentiMPHv2 plasmid prep?

[CB]

Hi, 

Has anyone else has a problem with low yields for lentiSAMv2 and lentiMPHv2? I got these plasmids from addgene along with 10 other plasmids which all grew up fine and I sequence confirmed them. However, SAM and MPH grew slowly when I plated them out on LB /carbenicillin. I have tried maxi-prep (using a starter culture) and mini-preps. Both were grown between 14-16 hours. I have high yields for lentiCRISPRv2 and every other lenti plasmid I work with. I'm surprised because both are high copy number. Unless there is too much plasmid and it is causing a problem with the recovery. The broth is turbid and pellet is expected size so I have bacteria just low plasmid yield. Any ideas?

Thanks, 

CB

[Julia Joung]

Posting the email thread here in case anyone else runs into the same issue:

Hi Carter,

Great to hear that the SAMv2 and MPHv2 plasmids checked out. It is interesting that they grow better in Stbl3 cells, because we have only used Stbl3 cells for our plasmid preps and were not aware that the NEB recombination deficient competent cells from Addgene do not produce as much plasmid.

Hope these plasmids will be helpful for your research!

Best,

Julia

On Sun, Apr 23, 2017 at 10:04 AM, Carter Barger wrote:

Hi Julia, 

Just to give you an update, I sequenced SAMv2 and MPHv2 along with performing diagnostic digests. The plasmids check out. They also grow much better after I transformed them into Stbl3 cells. I think addgene used NEB recombination deficient competent cells. 

Thanks,

Carter

On Thu, Apr 20, 2017 at 10:38 AM, Julia Joung wrote:

It's very strange that you are getting 3 bands instead of 2 - I would recommend targeted sequencing with an EF1a primer and contacting Addgene if the results do not align to the plasmid.

For minis, I usually do 3mL of TB for 24h and prep using the Qiagen kit. For midis, I do 4-6h of a 3mL TB starter culture, followed by expansion to 2x25ml TB for ~20h and prep using the Machery Nagel midi kit. I have cloned into these plasmids extensively, and have not seen this issue before.

On Thu, Apr 20, 2017 at 11:26 AM, Carter Barger wrote:

Thanks for the response. I have done the maxi prep once. However,the minipreps were done twice each time using a colony from a fresh plate streaked out from the addgene stock vial. Like I said, I grow a lot of lenti plasmid and I have done extensive cloning with very large plasmids but I have never had this issue. Maybe I will contact addgene. I also digested MPHv2 with EcoRI and Bsp1407I. It should show two bands but I am getting three bands. This was reproducible using independent preps streaked out from the stock vial. I am doing a more extensive diagnostic digest and targeted sequencing now. 

Are you growing them 12-16 hours and using a standard mini or maxiprep kit?

On Thu, Apr 20, 2017 at 10:10 AM, Julia Joung wrote:

Hi,

I have not encountered this problem when I grow lentiSAMv2 and lentiMPHv2 plasmids. For me, they usually produce much higher plasmid yield (ng) than other plasmids because they are larger, so I would recommend reprepping to double check that the there were no issues with the initial prep.

If anyone else encounters this problem, please contact Addgene and ask them to double check these plasmids.

Best,

Julia