Alpha Lactabulin notebook
2/2/23: designing Twist alpha-lactalbumin (a-lac) gene 2/14/23: First golden gate cloning 13.6 uL water 2 ul Ligase buffer 1 uL AmpBC backbone 1.4 uL a-lac gene 0.75 uL BsaI 1.25 uL ligase 2/20/23: performing E.Coli transformation and plating 2/21/23: plate did not grow overnight. Redoing the same golden gate procedure from the 14th but thermocycling for 18hrs instead of 1hr 2/22/23: transforming and plating 2/27/23: picked three white colonie for grow up 3/1/23: did mini preps to isolate the DNA 3/2/23: digest and gel Digest: 13 uL water 2 uL buffer 3 uL DNA 0.5 uL Oligo 0.5 uL AarI Gel:
3/9/23: DNA sent for sequencing 3/14/23: sequencing results aligned perfectly! Now adding promoter, Gmubi, and terminator, hstp. 3/20/23: Doing overnight grow up of glycerol stocks of the promoter and terminator 3/25/23: Doing a mini prep of the promoter and terminator 4/5/23: doing a 18hr golden gate 10.82uL water 2 uL ligase buffer 0.6 uL ChlorAD-B backbone 0.3 Gmubi 2 uL a-lac 1.75 uL hspt 0.75 PaqC1 0.5 uL restriction enzyme activator 1.25 uL ligase 4/6/23; transforming and plating 4/7/23: plate looks good grabbing white colonies for grow ups
4/11/23: Minipreps of the three grow ups 4/12/23: digest and gel electrophoresis Digest: 14 uL water 2 uL buffer 3 uL DNA 0.5 uL BsaI 0.5 uL BaeI Thermocycle
Not seeing three separate bands we are looking for. 4/17/23: Redoing the golden gate but increasing concentrations 8.2 uL water 2 uL ligase buffer 1.3 uL ChlorA DB 3 uL a-lac 2.75 uL HSPt 0.75 uL PaqC1 0.75 uL activator 1.25 uL ligase 4/19/23: transfecting and plating 4/20/23: The plates have all blue conlines and no white colonies 5/15/23:Redoing golden gate but increasing inserts but not the backbone concentrations 6.25 uL water 2 uL ligase buffer 0.6 uL ChlorA DB 1 uL Gmubi 4 uL a-lac 3.5 uL HSPt 0.75 uL PaqC1 0.75 uL Activator 1.25 uL ligase 5/17/23: transforming and plating 5/18/13: the plates did not grow any colonies 5/26/23: Not sure why the plates are not growing. Performing digest of ChlorA DB, Gmubi, and HSPt to see if they have the right DNA
All bands look as expected so now will redo all the math for the golden gate procedure as the inserts do not seem to be the problem 5/29/23: new golden gate 7 uL water 2 uL ligase buffer 0.6 ChlorA DB 2.7 Gmubi 2 uL a-lac 3.2 uL HSPt 0.75 uL PaqC1 0.5 uL activator 1.25 ligase Did a second golden gate where I doubled the volume of HSPt, Gmubi, and a-lac. 5/30/23: transformation and plating 5/31/23: first golden gate plate had two white colonies I picked for grow ups
6/2/23: mini preps, digest, and gel electrophoresis Did a double restriction digest because BsaI cuts two fragments of very similar lengths Digest: 14 uL water 2 uL buffer 3 uL DNA 0.5 uL BsaI 0,5 uL BaeI Thermocycle Gel electrophoresis: lane 1 ladder lane 2 sample 1 just BsaI Lane 3 sample 1 just BsaI Lane 4 sample 2 just BsaI Lane 5 sample 2 just Bsal Lane 6 sample 1 just BaeI Lane 7 sample 1 just BaeI Lane 8 uncut sample 1 Lane 9 uncut sample 2 lane 10 ladder bottom lanes: lane 1 ladder lane 2 sample 2 just BaeI Lane 3 sample 2 just BaeI Lane 4 sample 1 uncut Lane 5 sample 2 uncut
Picked colonies for an overnight grow up 7/18/23: Digest and gel electrophoresis Digest: 14 uL water 2 uL buffer 3 uL DNA 1 uL PSTI Thermocycle gel key: Expected
First three lanes are showing the correct band lengths 2 first lanes sent off for sequencing 8/8/23: all sequences aligned correctly! 8/25/23: Found out from other experiments the RUBY marker is not working as desired so will be now using a GUS marker and redoing the last plasmid golden gate with GUS in place of RUBY 10/5/23: GUS plasmid was constructed by a lab Technician and is now ready to go into the last backbone Golden gate 11.3 uL water 2 uL ligase buffer 1.2 uL pBEHA2 backbone 1.2 uL GUS 2.3 uL Chlor A DB/a-lax/gmubi/HSPt 0.75 uL BsaI 1.25 uL ligase 10/6/23: transformation and mini prep 10/10/23: digest and gel electrophoresis Digest: 14.5 uL water 2 uL buffer 3 uL DNA 0.5 uL BsaI Thermocycle Gel electrophoresis
Can’t see the lower band so will do a digest with a different restriction enzyme to get cut sites that are easier to visualize on a gel
Beta Lactoglobulin notebook
DNA shipment and processing: We received the TWIST order this weekend. Planning on transfecting the Bglobin DNA into NEB chemically competent cells. DNA is stored in box label: B-Lactoglobulin 1000ng DNA was diluted with 20ul H20 for a concentration of 20ng/ul Initial GoldenGate: 2/13/2023 Bglobulin insert (30ng goal @ 50ng/ul)- 1ul Amp B BC Plasmid(70ng goal at 140ng/ul)- .5ul T4 Ligase buffer (10x)- 2ul T4 Ligase- 1.25ul BsaI- .75ul H20- 14.5ul Total Volume- 20 ul 60 minute Golden Gate ran, store in Bglobulin box. Cell transformation: Date performed: 02/15 Changes in yellow Colony selection: Plates grew well from 2/15-2/16 with many white colonies and no blue colonies. 4 colonis were picked (2 from each plate) and grown out overnight (2/16-2/17) On 2/17 grow outs were drained and placed in freezer. Initial Miniprep: 2/20: Dna was miniprepped Dna Digestion for insert conformation: 02/27/2023 Dna was sigested with PAQC1 13ul H20 2ul Cut Smart 3ul DNA .5ul PAQC1 .5ul Activator --------------- 20 ul total 60 min GG Gel For Confirmation: Confirmed.. DNA Sequencing Results: Perfect, both BG 2,1 and BG 1,1 contain no errors Promoter/ Terminator Golden Gate Planning: A- Glycine Max Ubiquitin promoter - B - Beta Lactoglobulin - C - Arabadopsis therm HSP Terminator- D into A- XX -D-B Plasmid PAQC1 is used to open primary assembly Golden Gate- Preformed on 3/13/23 : Chlor A-DB- 55ng/ul stock- .75ul GMubi- 104ng/ul stock- .5 ul Beta Lactoglobulin (BG 2,1)- 60 ng/ul- 1ul Arabdopsis Term- 85 ng/ul Cell Transformation Chlor ADB with B globulin and promoter/term: Date performed: 03/14/2023 Notes: Used 2 5ul aloquats of the same cells, i am planning on making 4 plates 2 from each. Pro- Bg- Term grow outs: Went pretty well, not a lot of colonies but there is atleast 1 left on each plate. Mini Prep concentrations: 1.1 100 ng/ul 1.2 100 ng/ul 1.3 150 ng/ul 2.1 150 ng/ul Pro- Beta globulin- Term confirmatory gel: Virtual Digest: Real BsaI Digest: BG assembly + Chlor BD Ruby Planning plSUK backbone- .05 picomolar = 150 nanograms - 1.75 ul Betaglobulin assembly - 1,500 Bp- .1 picomolar= 97.5 nanograms- .75 ul ** used Betaglobulin - promoter/ Term assembly 2.1 from plate (A1-2) colony #1 ** Ruby - 6000 Bp- .1 picomolar = 390 nanograms- 1.75 ul T4 Ligase buffer (10x)- 2ul T4 Ligase- 1.25ul BsaI- .75ul H20- 11.75 ul Total Volume- 20 ul T4 Ligase buffer (10x)- 2ul T4 Ligase- 1.25ul BsaI- .75ul PaqcI - .5 ul PaqcI activator - .5 ul H20- 12.4 Total Volume- 20 ul 7/12/23 Golden Gate Planning for Recovered Lactoferrin to go into simple ( AmpB A-B) AmpB A-B (@97ng/ul) - 85ng required = .85ul Lactoferrin Recovered 2 (@80 ng/ul) - 200 ul required = 2.75 ul BsaI- .75ul T4 Ligase Buffer (10x) - 2ul T4 Ligase - 1.25ul H20- 12.4 ul Total Volume- 20ul 7/26/2023 Betaglobulin-PBEHA and Lactoferrin-Chlor-Adb transformation LB Agar Plate recipe: Kanamycin (Per 20ml agar plate) Xgal- 40ul IPTG- 100x- 200ul Iptg Kanna - 20ul LB Agar Plate recipe: Chlor (Per 20ml agar plate) Xgal- 40ul IPTG- 100x- 200ul Iptg Chlor- 20ul 10/6/2023 Betaglobulin-PBEHA with GUS marker Golden Gate Golden Gate Recipe pBEHAbackbone- .05 picomolar = 220nanograms (@ 250 ng/ul)= - .75 ul Betaglobulin assembly - 1,500 Bp- .1 picomolar= 82.5 ng @ ( 150ng/ul)= .75 ** used Betaglobulin - promoter/ Term assembly 2.1 from plate (A1-2) colony #1 ** GUS marker- .75 ul T4 Ligase buffer (10x)- 2ul T4 Ligase- 1.25ul BsaI- .75ul PaqcI - .5 ul PaqcI activator - .5 ul H20- 12.4 Total Volume- 20 ul Mini Prep 10/8/23 Prepped with Quiagen Mini-prep. Miniprep was 50ul at 200ng/ul.