Gibson assembly reagent prep and VR fragment validation #

Gibson assembly reagent prep #

Prepped new Gibson assembly reagents; 5x isothermal buffer and 1.33x master mix according to the Gibson assembly recipe spreadsheet.

5x isothermal buffer #

Gibson 1.33x master mix #

BglII digestion followed by PCR product validation #

Testing that BglII digestion followed by PCR amplification from 10/1/21 are the correct lengths by using VR-31 PCR product as a loading control. VR-31 was was supplied directly as a fragment and so PCR product should be exactly the correct length. All samples were composed of the reagents below.

• 3 ul PCR product
• 8 ul npH20
• 3 ul purple loading dye

Loaded all samples onto 0.8% 1x TAE agarose gel with 2 ul EtBr added to both the gel and 20 ul EtBr in the 1x TAE running buffer. Lane layout is shown in the table below.

Overall bands look like they are in the right place and so I moved on with the agarose gel extraction.

BglII digestion followed by PCR AGE #

Started gel with all BglII -> PCR samples for agarose gel extraction. Made the gel with 100 ml 0.8 agarose with 4 ul EtBr using 1x TAE for both the agarose gel and the running buffer. Added 40 ul EtBr to the running buffer. Ran gel at 120 V for 45 mins on undergrad bench. After running gel extracted VR insert bands using tin foil method on the trans-illuminator. Extracted DNA from bands using freeze and squeeze protocol.

Agarose extraction yields #

Agarose gel extraction product gel #

Given the high yield of the extractions I went ahead and ran the extracted products out on another gel. I loaded ~500 ng of each sample along with 3 ul purple loading dye and 8 ul H20 onto 0.8% 1x TAE agarose gel. Run the gel for 45 mins at 120V. Lane layout is below.

All bands look clean and are at the correct height (VR-30) loading control in lane 1. Moving forward with Gibson assembly of these fragments into pFC9.