Troubles in cloning my custom library

I have some troubles in clone my custom library, but I'm not sure what's wrong.

My custom library contains 12000 sgRNAs, which is small. but when we performed the NGS after plasmids extraction, it showed that only 1000 sgRNA was detected, and among the 1000 sgRNA, 300 sgRNA was abundant while others was only 1~2 copy.

1. We performed 26 cycle in PCR amplification of pooled oligo library with NEBnext, the protocol suggest less than 20 cycles, but the PCR product is weak when use 20cycle. I wonder if the 26 cycle indroduced the biases?

2. The protocol suggest "After the reaction is complete, pool the PCR reactions and purify the PCR product using the QIAquick PCR Purification Kit, then Run the PCR-purified oligo library from Step 5 on a gel , after that Gel-extract the purified PCR product using the QIAquick Gel Extraction Kit" but we performed gel-extract the PCR product without do PCR Purification, does it matters?

3.Gibson assembly was done according the protocol.

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Fuchsia Kelpieabout 2 years ago

To answer your questions:

1. Yes 26 cycles will introduce more bias, but unlikely to be the root cause of your observation.

2. Sometimes without PCR extraction, the PCR product can run more like a smear instead of a clear band, so I would recommend doing the PCR purification before gel extraction.

From here I would actually recommend that you sequence the PCR product of your synthesized oligo pool directly, by performing another PCR to add the Illumina adapters - this will help you narrow down whether the problem occurred before or after Gibson assembly. I have had bad oligo syntheses before that had low fidelity and high bias, so it is worth checking if your synthesized oligo pool is good.

Fuchsia Kelpieabout 2 years ago

Yes that should be fine.

Sienna Faunabout 2 years ago

I reanalysis the step during the library constructing, I think the electroporation efficiency didn’t reach 20 times more colonies per electroporation in the sgRNA library condition as compared with the control Gibson reaction. Do you think the low transformation efficiency may cause this?

I did a pre-experiment this week, I performed 20 cycle in PCR amplification of pooled oligo library with NEBnext this time, and get enough oligo.

chemical cell was used instead of Electroporate cells in this per-experiment. Gibson assembly and CPEC were both used, after the reaction was complete, we Purify and concentrate the sgRNA library, the plasmid pellet was resuspend in 5ul of ddh20, the concentration were measured by Nanodrop and attached below, but the 260/230 is low than expect(>1.8).

Gibson control: 230.8ng/ul,A260/A280=1.89,A260/A230=1.11

Gibson library: 184.5ng/ul,A260/A280=1.89,A260/A230=1.19

CPEC control: 322.3ng/ul,A260/A280=1.80,A260/A230=1.2

CPEC library: 210.1ng/ul,A260/A280=1.80,A260/A230=1.22

1ul of the eluent was transformed into the chemical cells,

Then 10% (100 µl/1ml) of the cells were plated on a prewarmed standard LB agar plate,after 14h incubation,the colony numbers were counted and the results were attached below:

Gibson control: 77

Gibson library: 594

CPEC control:42

CPEC library:270

The fold change between library condition was only 7 times more colonies than control,

How can I improve the efficiency to reach 20 more times? I think the low efficiency may be the reason that I failed last time.

Fuchsia Kelpieabout 2 years ago

It does look like you are having a lot of background, that is probably independent of the nanodrop QC metrics. I would suggest varying the amount of sgRNA insert relative to the plasmid for the Gibson (up to a 2:1 ratio of insert:vector), to see if you get higher efficiencies.

Sienna Faunabout 2 years ago

I want to confirm that the ratio you suggest is molar ratio or mass ratio? According to the protocol, the mass ratio is 50ng:330ng(insert mass:vector mass), do you mean that I should reduce the vector, and use 50ng:25ng(insert mass:vector mass)? or 2:1 ratio of insert:vector is the molar ratio?

Thanks for your help!

Fuchsia Kelpieabout 2 years ago

Ah sorry I was referring to the mass ratio - maybe up to 330ng:330ng (insert mass:vector mass) should be enough?

Sienna Faunabout 2 years ago

Thanks for your suggestion,I will tried next week!

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