Objective and summary:

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• Run ethidium bromide gel electrophoresis

• Note: ran with a 1.5% agarose gel 100 mV for 60 minutes

• review Qubit for these extractions on 20220531

Gel Electrophoresis

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Gel map is as follows:

• ‘raw’ refers to the samples run as direct aliquots of the DNA extraction using (attachd protocol here)

• ‘I’ refers to the Illumina DNA Prep protocol as suggested by manufacturers using all Illumina materials with 500 ng DNA samples (SOP here)[https://github.com/SamGurr/SamJGurr_Lab_Notebook/blob/master/_posts/2022-06-03-Illumina-DNA-Prep-SOP]

• ‘I+N’ refers to the Illumina DNA Prep protocol for tagmentation only alongside amplification and cleanup using KAPA HiFi ReadyStart Mix and Ampure Beads w/tris resuspension as recommended by Nina’s modified Baym protocol (SOP here)[https://github.com/SamGurr/SamJGurr_Lab_Notebook/blob/master/_posts/2022-06-03-Illumina-DNA-Prep-with-KAPA-and-Ampure-SOP]

  • note this modified Baym prtocol calls for samples between 1-10ng (ideally 2.5) whereas I ran these as 500 ng to directly compare to the full Illumina - we will see if using the same reagent volumes (i.e. KAPA and Ampure beads) presents any red flags

well	1	2	3	4	5	6	7	8
samp	L	L	raw 1	raw 2	1 I	2 I	1 I+N	2 I+N

Gel image

• we see strong high molecular weight bands for the raw and no bands for the post-library prep samples

• I read (here[https://support.illumina.com/bulletins/2016/05/library-quantification-and-quality-control-quick-reference-guide.html] that the proper quality check for libraries from Illumina DNA Prep (& previous Nextera reagnets) is Quantification via Qubit and Quality control with a BioAnalyzer

Qubit quanitification

• followed-up up with Qubit and yielded ‘Too Low’ - no DNA present

What happened / what can I change?

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Note: Since library prep was run two different ways, it appears that the culprit lies in what was common between both SOPs (at least this is a good first step..) Common between both SOPs is the tagmentation and post-tagmentation clean-up and the use of the thermocycler.

• Tagment wash buffer

  • What happened? – A tip on the Illumina site emphasises that the Tagment Wash Buffer must be pipetted directly onto the beads where I simply added the wash buffer to fill during the wash steps.

  • What can I change? – Adding directly should disperse the beads from the magnet breifly, whether this will do the trick? Time will tell!

• PCR and thermocycler step(s)

  • What happened? – After reading through the protocol, all steps were completed correctly (or my best try in doing so). The only hiccup was in loading the thermocycler with the PCR plate - I noticed the plastic plate was quite warm after each step in the thermocycler, this was a mistake.

  • What can I change? – Next library prep attempt, load pcr tubes/96 well plate directly on the wells in the thermocycler to allow proper annealing temp and cooling for PCR amplification. These tubes/wells have thin walls created jsut for this purpose - improper loading of your sample to the thermocycler (i.e. within a loading plate) does not allow the tube/sample to be properly inserted in the thermocycler leading to error in temperature change