Catherine Channell - Dahlquist Lab

Catherine Channell

Spring 2021

Week 3

1/29

• editing tasks: https://openwetware.org/wiki/BIOL368/F20:Week_1
• check out new database: https://www.ncbi.nlm.nih.gov/snp/docs/gsr/alfa/

Week 4

2/2

• complete tutorial assignment on openwetware

2.5

Summer 2021

Week 1

5/19 @1-2 PM: Meeting w/Dr. Dahlquist

• OpenWetWare Tasks

Week 2

5/26 @1-2 PM: Meeting w/Dr. Dahlquist

• Add image, pdf, template, and fix Service and Leadership section

5/26 @2:30-6:30 PM: Meeting w/Claire

• Article Searches 2020-2021 using 'lactase persistence frequency' at https://pubmed.ncbi.nlm.nih.gov/
  • Anguita-Ruiz, A., Aguilera, C. M., & Gil, Á. (2020). Genetics of Lactose Intolerance: An Updated Review and Online Interactive World Maps of Phenotype and Genotype Frequencies. Nutrients, 12(9). https://doi.org/10.3390/nu12092689
    • Created two online interactive resources, which constitute an upgrade over previously published static world maps, and allow users a personalized data exploration, while at the same time accessing complete reports by population or ethnicity
  • Fung, M., Xue, X., & Szilagyi, A. (2020). Estimating Lactase Nonpersistence Distributions in the Multi-Ethnic Canadian Demographic: A Population-Based Study. Journal of the Canadian Association of Gastroenterology, 3(3), 103–110. https://doi.org/10.1093/jcag/gwy068
    • Used the latest 2016 population census into our sampled population and used Storhaug data of global LNP prevalence to derive LNP estimation rates for the Canadian demographic
  • Hollfelder, N., Babiker, H., Granehäll, L., Schlebusch, C. M., & Jakobsson, M. (2021). The genetic variation of lactase persistence alleles in Sudan and South Sudan. Genome biology and evolution, evab065. Advance online publication. https://doi.org/10.1093/gbe/evab065
    • Research paper that collected/sequenced frequencies of genetic variants linked to LP in Sudanese and South Sudanese populations
  • Dewiasty, E., Setiati, S., Agustina, R., Roosheroe, A. G., Abdullah, M., Istanti, R., & de Groot, L. C. (2021). Prevalence of lactose intolerance and nutrients intake in an older population regarded as lactase non-persistent. Clinical nutrition ESPEN, 43, 317–321. https://doi.org/10.1016/j.clnesp.2021.03.033
    • data on the prevalence of lactose intolerance and dietary intakes in Indonesian older outpatients based on a hydrogen breath test
• no additional articles using 'lactase persistence population' at https://pubmed.ncbi.nlm.nih.gov/
• no additional articles using 'lactase persistence' at https://pubmed.ncbi.nlm.nih.gov/
• articles using 'lactase persistence' at http://apps.webofknowledge.com.electra.lmu.edu/WOS_GeneralSearch_input.do?product=WOS&search_mode=GeneralSearch&SID=8Fzg1zagUWiBD6DBnTk&preferencesSaved=
  • Jacobs, E. T., Foote, J. A., Kohler, L. N., Skiba, M. B., & Thomson, C. A. (2020). Re-examination of dairy as a single commodity in US dietary guidance. Nutrition Reviews, 78(3), 225–234. https://doi.org/10.1093/nutrit/nuz093
    • Following Canada's removal of lactose as a separate food group for national dietary guidelines, a suggestion on why the US should think about doing the same
• no additional articles using 'lactase persistence' at https://search-sciencemag-org.electra.lmu.edu/?searchTerm=lactase%20persistence&order=tfidf&limit=textFields&pageSize=10&startDate=2020-01-01&endDate=2021-06-01&&
• articles using 'lactase persistence frequency' at https://scholar.google.com/
  • Борозенець, В. В., Бабалян, В. О., & Федота, O. M. (2020). Genetiс aspects of lactase persistence in different ethnic groups. Фактори експериментальної еволюції організмів, 27, 185-189.
    • lactase persistence frequencies in different European ethnic groups (Ukrainian, Indian, Nigerian, Israeli, Egyptian, Palestinian, Turkish, Jordanian students (n=361) and their relatives of the first degree of kinship (n=413))
  • Ramadan, M., Eid, A. M., Seliem, N. M., & Shokr, H. A. (2020). Assessment of lactose intolerance in adult Egyptian patients with dyspeptic symptoms using lactase enzyme assay in duodenal biopsy. The Scientific Journal of Al-Azhar Medical Faculty, Girls, 4(2), 300.
    • Study of the severity of lactose-intolerance symptoms in Egyptian patients
  • Ojeda-Granados, C., Panduro, A., Rivera-Iñiguez, I., Sepúlveda-Villegas, M., & Roman, S. (2020). A Regionalized Genome-Based Mexican Diet Improves Anthropometric and Metabolic Parameters in Subjects at Risk for Obesity-Related Chronic Diseases. Nutrients, 12(3), 645.
    • Provides an implemented example of how taking into account LNP can result in a healthy, balanced diet
  • Mørk, M., Andersen, S. L., Pedersen, I. S., Ernst, A., Lykkeboe, S., & Krarup, H. B. (2020). Use of genetic testing for hypolactasia trait in the North Denmark region. Scandinavian Journal of Gastroenterology, 55(9), 1012-1018.
    • Denmark frequencies of LP and LNP https://www.tandfonline.com/doi/abs/10.1080/00365521.2020.1800079
  • Martínez Rodríguez, M., Samaniego-Vaesken, M., & Alonso-Aperte, E. (2021). A New Food Composition Database of Lactose-Free Products Commercialized in Spain: Differences in Nutritional Composition as Compared to Traditional Products. Foods, 10(4), 851.
    • support for viability of lactose-free alternatives https://www.mdpi.com/2304-8158/10/4/851

Week 3

6/2 @1-2 PM: Meeting w/Dr. Dahlquist

• Read Review Article on World Maps
• Read Segurel, L., Guarino-Vignon, P., Marchi, N., Lafosse, S., Laurent, R., Bon, C., Fabre, A., Hegay, T., & Heyer, E. (2020). Why and when was lactase persistence selected for? Insights from Central Asian herders and ancient DNA. PLOS Biology, 18(6), e3000742. [1]
• Journal club on Canadian study
  • 1+ intro slides
  • titles as messages
  • figure and tables each get their own slide
  • conclusion

6/2 @2-6 PM: Meeting w/Claire

Week 4

6/2 @1-2 PM: Meeting w/Dr. Dahlquist

• Read Review Article on World Maps
• Read Storhaug et. al

6/2 @2-6 PM: Meeting w/Claire

Week 5

6/16 @1-2 PM: Meeting w/Dr. Dahlquist

• approach for paper will be review of genetic information available for U.S. on different databases
• start with Ensemble and 13910
  1. individuals in pop. sampled, where they are from/ethnicity, & traceable data needed in order to avoid duplication of studies

6/17 @2:30-6:30 PM: Meeting w/Claire

• Smith et. al (2016) to 2002

Week 6

6/23 @1-2 PM: Meeting w/Dr. Dahlquist

• Send dbSNP email about criterion for 89 publications
• Finish checking publications
• Add reason why excluded
• General Summary of Data Once all Sorted Through

Week 7

6/30 @1-2 PM: Meeting w/Dr. Dahlquist

• 2-5 page summary of work to date

Week 8

7/7 @1-2 PM: Meeting w/Dr. Dahlquist

Fall 2021

Week 1

8/31: Micropipetting Technique; Evaluating Accuracy and Precision NB1 p46-50

• Purpose:

Week 2

9/7: Micropipetting Technique; Evaluating Accuracy and Precision Cont. NB1 p50-52

• Purpose:

Week 3

9/14: Micropipetting Technique; Evaluating Accuracy and Precision Cont. NB1 p52-57

• Purpose:

Week 4

9/21: Restriction Digest of Lambda (λ) DNA NB1 p57

• Purpose:

Week 5

9/21: Restriction Digest of Lambda (λ) DNA NB1 p58-60

• Purpose:

Spring 2022

Week 15

4/7 RD gel of 926, 546, 235, 791#1, 956#1 NB1 p93

• Purpose: Analysis & RD gel of purified amplified samples by Claire, Erykah, & Liz

RD gel Protocol:

• Poured 1:29pm, start 2:22 pm

Fall 2023

with Odoba Okwuosa

Week 1

8/31 3:30-5:30 pm: PCR of Summer Samples 642, 412, 548, 617, 718 NB1 p94-95

• Purpose: PCR summer '22 collected LMU Samples

Week 2

9/5

• Labor Day

9/7 3:30-5:30 pm: PCR gel of 642, 412, 548, 617, 718 and PCR of 695, 972, 226, 599, 742, 626 NB1 p95-96

• Purpose: PCR summer '22 collected LMU Samples

PCR gel protocol: start 4:05 pm

• remove 5 μL of PCR product -> microcentrifuge tube + 1 μL 6X loading dye, mix & spin
• load 10 μL 1kb DNA ladder to L-most lane
• load other samples into wells
• run @120V until bromophenol blue ->2-3cm
  • expected product=448 bp
  • Dilute plasmids 1:10; move neg. control to R-most lane to avoid crossover; successful yield of PCR product

Week 3

9/12 3:30-5:30 pm: PCR gel of 6/16 collected samples 695, 972, 226, 599, 742, 626 and PCR of 6/21 collected samples 711, 795, 459, 396, 877, 740 NB2 p71-72

• Purpose: PCR summer '22 collected LMU Samples

9/14 3:30-5:30 pm: PCR gel of 695, 972, 226, 599, 742, 626 and PCR gel of 711, 795, 459, 396, 877, 740 NB2 p72-73

• Purpose: PCR summer '22 collected LMU Samples

Week 4

9/19 3:30-5:30 pm: PCR of 183, 682, 641, 294, 699 NB2, p74

• Purpose: PCR summer '22 collected LMU Samples

9/21 3:30-5:30 pm: PCR gel 183, 682, 641, 294, 699 NB2 p74-75

• Purpose: PCR summer '22 collected LMU Samples

Protocol:

• • No PCR product observed

Week 5

9/26 3:30-5:30 pm: PCR 695, 972, 226, 599, 742, 626 NB2 p76

• Purpose: PCR summer '22 collected LMU Samples

9/28 3:30-5:30 pm: PCR gel 695, 972, 226, 599, 742, 626 NB2 p76-77

• Purpose: PCR summer '22 collected LMU Samples

Week 6

10/3 3:30-5:30 pm: Re-do PCR Gel w/10 template/sample to troubleshoot NB2 p77-78

• Purpose: PCR summer '22 collected LMU Samples

10/5 3:30-5:30 pm: PCR gel 711, 795, 459, 396, 877, 740, 183 and PCR 250, 508, 989, 682, 641, 294, 699 NB2 p78-79

• Purpose: PCR summer '22 collected LMU Samples

Week 7

10/10 3:30-5:30 pm: PCR gel 250, 508, 989, 682, 641, 294, 699 NB2 p79-80

• Purpose:

PCR gel protocol: start 3:56 pm

• remove 5
  • Re-run: 711, 795, 459, 396, 250, 508, 989, 682, 641, 294, 699

10/12 3:30-5:30 pm: PCR 711, 795, 459, 396 NB2 p80-81

• Purpose: PCR summer '22 collected LMU Samples

Week 8

10/17 3:30-5:30 pm: PCR gel 711, 795, 459, 396 and PCR 250, 508, 989, 682 NB2 p81-82

• Purpose: PCR summer '22 collected LMU Samples

10/19 3:30-5:30 pm: PCR gel 250, 508, 989, 682 and PCR 641, 294, 699 NB2 p82-83

• Purpose: PCR summer '22 collected LMU Samples

Week 9

10/24 3:30-5:30 pm: PCR & PCR gel 641, 294, 699 and Summary & Organization of PCR Results NB2 p84-85

• Purpose: PCR summer '22 collected LMU Samples

10/26 3:30-5:30 pm: Purification CC, CT, CT, and Hela NB2 p87

• Purpose: Check for Hela degradation and plasmid purity

Week 10

10/31 3:30-5:30 pm: RD and RD gel lambda, CC, CT, CT, and Hela NB2 p87

• Purpose: Check for Hela degradation and plasmid purity

11/2 3:30-5:30 pm:

Week 11

11/7 3:30-5:30 pm: Purification and RD 183, 226, 250 NB2 p88

• Purpose: Purification & RD of summer '22 collected LMU Samples

11/9 3:30-5:30 pm: RD gel 183, 226, 250 and Purification 396, 412, 459, 548

• Purpose: Purification & RD of summer '22 collected LMU Samples

Week 12

11/14 3:30-5:30 pm: RD 396, 412, 459, 548 NB2 p90

• Purpose: Purification & RD of summer '22 collected LMU Samples

11/16 3:30-5:30 pm: RD Gel 396, 412, 459, 548 NB2 p90-91

• Purpose: Purification & RD of summer '22 collected LMU Samples

Week 13

11/21 3:30-5:30 pm: Purification 599, 617, 626, 641 NB2 p91

• Purpose: Purification & RD of summer '22 collected LMU Samples

Week 14

11/28 3:30-5:30 pm: RD 599, 617, 626, 641 NB2 p92

• Purpose: Purification & RD of summer '22 collected LMU Samples

Restriction Digest w/BsmFI Protocol:

• λ control: 4 μL (0.1 μg/mL) λ DNA + 6 μLsterile MilliQ water
• 10 μL purified PCR product -> fresh 0.5 μL tube
• master mix on ice w/BsmFI (NEBio #R0572S) 9.5X
  • sterile MilliQ water 66.5 μL
  • 10X Cut Smart buffer 19 μL
  • MIX & SPIN also, start thermocycler ealy b/c of 4 min preheat on lid -> pause until ready
  • BsmFI (2 units/μL) 9.5 μL
• add 10 μL MM to each tube -> mix & spin
• incubate @65°C for 60 min, @80°C for 10 min "BSMFI" on small thermocycler
• Pour 1.5% agarose gel w/1X TAE Buffer & [0.5 μg/mL] EtBr start 4pm, poured 4:10pm
  • LARGE=1.5g agarose, 10 mL 10X TAE + 90 mL water, 100 μL EtBr

11/30 3:30-5:30 pm: RD Gel 599, 617, 626, 641 and Purification of 642, 682, 695, 711, 718, NB2 p92-93

• Purpose: Purification & RD of summer '22 collected LMU Samples

RD Gel Protocol: start 3:55 pm

• add 4 μL of 6X loading dye to cut & 2 μL to uncut
• load 10 μL Purple 100 bp DNA ladder (NEB #N0551S) in L most lane
• load samples onto gel (uncut L, cut R) w/λ in R-most
• run @120V
  • CC(351, 97), TT(253, 98/97), CT (351, 253, 98/97 bp)
  • 599, 617, 626, 641 appear ___

Purification Protocol:

• use DNA Clean & Concentrator Kit (Zymo D4014; Genessee 11-303C)
• In a 1.5 mL microcentrifuge tube, add 5 volume DNA binding buffer, mix & spin
  • 642, 695, 718(45 μL -> 225 μL) and 682, 711 (90 μL -> 450 μL)
• mixture --> zymo-spin column in collection tube --> spin 30 sec, 10, 000X G
• Discard flow-thru and reinsert
• Repeat wash step & spin AGAIN (2 times w/o adding ligand) 30 sec, 10, 000 X G
• transfer column to clean 1.5 mL tube w/cap cut off
• add 20 μL DNA elution buffer to matric
• incubate @room temp for 1 min
• spin 30 sec, 10, 000 X G then record volume of elute as you transfer --> 1.5 mL tube w/lid
  • CC=18.6 μL
  • TT=18.7 μL
  • CT=18.4 μL
  • Hela=18.4 μL
  • 642=18.4 μL
  • 682=18.2 μL
  • 695=18.3 μL
  • 711=18.2 μL
  • 718=18.4 μL

Week 15

12/5 3:30-5:30pm: Restriction Digest w/BsmFI 642, 682, 695, 711, 718

• Purpose:

Restriction Digest w/BsmFI Protocol:

• λ control: 4 μL (0.1 μg/mL) λ DNA + 6 μLsterile MilliQ water
• 10 μL purified PCR product -> fresh 0.5 μL tube
• master mix on ice w/BsmFI (NEBio #R0572S) 10.5X
  • sterile MilliQ water 73.5 μL
  • 10X Cut Smart buffer 21 μL
  • MIX & SPIN also, start thermocycler ealy b/c of 4 min preheat on lid -> pause until ready
  • BsmFI (2 units/μL) 10.5 μL
• add 10 μL MM to each tube -> mix & spin
• incubate @65°C for 60 min, @80°C for 10 min "BSMFI" on small thermocycler
• Pour 1.5% agarose gel w/1X TAE Buffer & [0.5 μg/mL] EtBr
  • LARGE=1.5g agarose, 10 mL 10X TAE + 90 mL water, 100 μL EtBr

12/7 3:30-5:30pm: RD Gel 642, 682, 695, 711, 718

• Purpose:

RD Gel Protocol:

• add 4 μL of 6X loading dye to cut & 2 μL to uncut
• load 10 μL Purple 100 bp DNA ladder (NEB #N0551S) in L most lane
• load samples onto gel (uncut L, cut R) w/λ in R-most
• run @120V
  • CC(351, 97), TT(253, 98/97), CT (351, 253, 98/97 bp)
  • 682 and 718 appear CC; 642, 695, and 711 and appear CT

Spring 2023

with Odoba Okwuosa

Week 2

1/18 1:40-5:40 pm: Purification & Restriction Digest of 740, 742, 795, NB3 p1

• Purpose: 2nd to last purification & RD of summer '22 collected LMU Samples (PCR done Fall 2022)

Purification Protocol:

• use DNA Clean & Concentrator Kit (Zymo D4014; Genessee 11-303C)
• In a 1.5 mL microcentrifuge tube, add 5 volume DNA binding buffer, mix & spin
• mixture --> zymo-spin column in collection tube --> spin 30 sec, 10, 000X G
• Discard flow-thru and reinsert
• Repeat wash step & spin AGAIN (2 times w/o adding ligand) 30 sec, 10, 000 X G
• transfer column to clean 1.5 mL tube w/cap cut off
• add 20 μL DNA elution buffer to matric
• incubate @room temp for 1 min
• spin 30 sec, 10, 000 X G then record volume of elute as you transfer --> 1.5 mL tube w/lid
  • CC=18.5 μL
  • TT=17.7 μL
  • CT=18.7 μL
  • 740=18.5 μL
  • 742=20.0 μL
  • 795=19.3 μL

Restriction Digest w/BsmFI Protocol: start 3:04 pm

• λ control: 4 μL (0.1 μg/mL) λ DNA + 6 μLsterile MilliQ water
• 10 μL purified PCR product -> fresh 0.5 μL tube
• master mix on ice w/BsmFI (NEBio #R0572S) 7.5X
  • sterile MilliQ water 52.5 μL
  • 10X Cut Smart buffer 15 μL
  • MIX & SPIN
  • BsmFI (2 units/μL) 7.5 μL
• add 10 μL to each tube -> mix & spin
• incubate @65°C for 60 min, @80°C for 10 min "BSMFI" on small thermocycler
• add 4 μL of 6X loading dye to cut & 2 μL to uncut

RD Gel Protocol: start 4:05 pm

• Pour 1.5% agarose gel w/1X TAE Buffer & [0.5 μg/mL] EtBr poured 3:13 pm
  • LARGE=1.5g agarose, 10 mL 10X TAE + 90 mL water, 100 μL EtBr
• load 10 μL Purple 100 bp DNA ladder (NEB #N0551S) in L most lane
• load samples onto gel (uncut L, cut R) w/λ in R-most
• run @120V
  • CC(351, 97), TT(253, 98/97), CT (351, 253, 98/97 bp)
  • All (740, 742, 795) appear to be CC

Week 3

1/25 1:40-5:40 pm: Purification & Restriction Digest of 877, 972, 989, NB3 p2-3

• Purpose: Last purification & RD of summer '22 collected LMU Samples (PCR done Fall 2022)

Purification Protocol:

• use DNA Clean & Concentrator Kit (Zymo D4014; Genessee 11-303C)
• In a 1.5 mL microcentrifuge tube, add 5 volume DNA binding buffer, mix & spin
  • 989(45 μL -> 225 μL) and 877, 972 (90 μL -> 450 μL)
• mixture --> zymo-spin column in collection tube --> spin 30 sec, 10, 000X G
• Discard flow-thru and reinsert
• Repeat wash step & spin AGAIN (2 times w/o adding ligand) 30 sec, 10, 000 X G
• transfer column to clean 1.5 mL tube w/cap cut off
• add 20 μL DNA elution buffer to matric
• incubate @room temp for 1 min
• spin 30 sec, 10, 000 X G then record volume of elute as you transfer --> 1.5 mL tube w/lid
  • CC=20.0 μL
  • TT=18.2 μL
  • CT=19.0 μL
  • Hela=degraded
  • 877=20.0 μL
  • 972=19.25 μL
  • 989=18.9 μL

Restriction Digest w/BsmFI Protocol: start 2:40 pm

• λ control: 4 μL (0.1 μg/mL) λ DNA + 6 μLsterile MilliQ water
• 10 μL purified PCR product -> fresh 0.5 μL tube
• master mix on ice w/BsmFI (NEBio #R0572S) 7.5X
  • sterile MilliQ water 52.5 μL
  • 10X Cut Smart buffer 15 μL
  • MIX & SPIN also, start thermocycler ealy b/c of 4 min preheat on lid -> pause until ready
  • BsmFI (2 units/μL) 7.5 μL
• add 10 μL to each tube -> mix & spin
• incubate @65°C for 60 min, @80°C for 10 min "BSMFI" on small thermocycler
• add 4 μL of 6X loading dye to cut & 2 μL to uncut

RD Gel Protocol: start 4:05 pm

• Pour 1.5% agarose gel w/1X TAE Buffer & [0.5 μg/mL] EtBr
  • LARGE=1.5g agarose, 10 mL 10X TAE + 90 mL water, 100 μL EtBr
• load 10 μL Purple 100 bp DNA ladder (NEB #N0551S) in L most lane
• load samples onto gel (uncut L, cut R) w/λ in R-most
• run @120V
  • CC(351, 97), TT(253, 98/97), CT (351, 253, 98/97 bp)
  • 877 and 972 appear CC; 989 is inconclusive

Week 4

2/1 1:40-5:40 pm:

Week 5

2/8 1:40-5:40 pm:

Week 6

2/15 1:40-5:40 pm:

Week 7

2/22 1:40-5:40 pm:

Week 8

3/1 1:40-5:40 pm:

• Following Week is Spring Break

Week 9

3/15 1:40-5:40 pm: Finalize and Print Poster for LMU URS on 3/17

Week 10

3/22 1:40-5:40pm: Create new figures based on self-reported lactose consumption from survey results

Week 11

3/29 1:40-5:40pm: Update Poster for 2023 WCBSURC by modifying discussion based on new figures

Week 12

Easter Break

Week 13

4/12 1:40-5:40 pm: Update OpenWetWare

Fall 2023

Week 1

8/31

• Lab Meeting to discuss new semester and welcome new lab members

Week 2

9/7

• Lab Meeting to catch new lab members up to speed on lactase persistence project

Week 3

9/14

• Pipetting exercise with Nathan

Week 4

9/21

• PCR Gel and Purification, 720 & 202
  • Purpose: To run PCR Gel on products from 720 & 202 and then continue data collection of LP frequencies at LMU
  • Protocol:
    • Run Gel:
      • Remove 5 uL PCR products --> microcentrifuge tubes & add 1 uL loading dye. Mix & Spin.
      • Load 10 uL 1 kb DNA ladder to L-most lane
      • Load other samples
      • Run at 120 V until bromophenol blue --> 2-3 cm
      • Expected products: 448 bp
      • START: 2:34 PM
      • STOP: 3:18 PM
        • Key:
        • 0=1kb ladder
        • 1=
        • 2=
        • 3=
        • 4=
        • 5=
        • 6=

Week 5

9/28

Week 6

10/5

Week 7

10/12

Week 8

10/19

Week 9

10/26

• Modified RD
  • Purpose: To

Week 10

11/2/23

• Purification without Plasmids
  • Purpose: Due to incomplete digest, but distinct bands of 10/26/23 modified digest & remaining concern about TT plasmid contamination, we are running a new PCR & RD without the plasmids
  • Protocol:
    • PCR gel was started by Odoba at 1: 42 pm
    • Lanes of PCR gel "PCR_2023-11-2_125msec.tif"
    • 1. H2O (Negative control)
    • 2. HeLa
    • 3. 720
    • 4. 202

Week 11

11/9/23

Week 12

11/16/23

Week 13

11/23/23

• Thanksgiving

Week 14

11/30/23

• QIAamp DNA Investigator Kit (50) Cat# 56504
• protocol Dahlquist:DNA Extraction from Human Hair

Spring 2024

with Odoba Okwuosa

Week 2

1/17 12:00-6:00 pm: QIAamp Test Run

• Purpose: To test run the new QIAamp forensic DNA hair extraction protocol for improved optimization

QIAamp Protocol: Dahlquist:DNA Extraction from Human Hair

• Prior steps done by Odoba & Naomi this morning
• Note: ATE + RNA mixture is only to be used twice, today is was used once, so it should be thrown out the next time it is used.

1. Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
   • If lysate remains, spin again at higher speed
2. Place the column in new collection tube. Discard the collection tube with flow-through
3. Add 500 μL of Buffer AW1 to the column.
   • Do not wet rim.
4. Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
5. Place the column in new collection tube. Discard the collection tube with flow-through.
6. Add 700 μL of Buffer AW2 to the column.
   • Do not wet rim.
7. Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
8. Place the column in new collection tube. Discard the collection tube with flow-through. Make sure that flow-through has not come in contact with the column. If it has, spin again.
9. Add 700 μL of ethanol (96-100%) to the column.
   • Do not wet rim.
10. Spin for 1 minute at 6,000 X g (8000 rpm) in the microcentrifuge.
11. Place the column in new collection tube. Discard the collection tube with flow-through.
12. Spin at full speed (15,000 X g; 14,000 rpm) for 3 min to dry the membrane completely.
    • It is important that this is completely dry!
13. Place the column in 1.5 mL microcentrifuge tube with cap cut off. Discard the collection tube with flow-through.
14. Open lid of QIAamp MinElute Column, and incubate at room temperature for 10 min or at 56°C for 3 min.
15. Apply 50 µl room temperature Buffer ATE or distilled water to the center of the membrane.
16. Close the lid and incubate at room temperature for 5 min
17. Centrifuge at full speed (20,000 x g; 14,000 rpm) for 1 min.
18. Carefully transfer eluate to a labeled 1.5 mL microcentrifuge tube with a lid. Use immediately in PCR or store at -20°C.

PCR Protocol

• Number 0.2 mL PCR tubes.
• Add 15 μL of PCR-quality water to each tube.
• Add 2.5 μL each of 10 μM Lac-13910-for-EM and Lac-13910-rev-EM primers.
• Add 5 μL template to each tube according to the list below.
  1. negative control, no template, use 5 μL sterile MilliQ water in its place
  2. CC plasmid positive control (1:200 dilution), 5 μL
  3. TT plasmid positive control (1:200 dilution), 5 μL
  4. CT mixed plasmid positive control (1:200 dilution), 5 μL
  5. HeLa genomic DNA (5 ng/μL), 5 μL
  6. samples 6-n, cheek cell or hair follicle lysate (5 μL)
• Flash spin tubes to bring templates to bottom of the tube.

PCR Master Mix Protocol

• Turn on the thermocycler.
  • Run "PHUSION" program (Bio-Rad S1000 thermocycler in FEA 263).
  • Once lid has reached 100°C and the block has reached 98°C, pause the program by pressing the "pause" button.
• Remove 2X Phusion Flash High Fidelity PCR Master Mix (Cat# F548S) from freezer and thaw on ice. Mix and flash spin (gently, do not vortex!)
• Add 25 μL of the 2X Phusion Master Mix to each tube. Flick to mix and flash spin.
• Place tubes in thermocycler and un-pause program by pressing the "pause" button again.

PCR Thermocycler Program: Started at 3:00 PM

Program name on Bio-Rad S1000 thermocycler in FEA 263 is "PHUSION"

1. 98°C 10 seconds
2. 98°C 01 seconds
3. 64°C 05 seconds
4. 72°C 15 seconds
5. repeat steps 2-4 for 35 cycles
6. 72°C 1 minute
7. hold at 6°C

PCR Gel: Poured at 3:30 PM

Pour 1% Small PCR Gel (total volume 50 mL): 0.5g agarose, 5 mL 10X TAE + 45 mL water, 50 μL EtBr

Run PCR Gel, started 4:37 PM

1. Remove 5 μL of PCR product and transfer to 1.5 mL microcentrifuge tubes.
2. Add 1 μL loading dye to each. Mix & Spin.
3. Add 10 μL 1 kb DNA ladder to the left-most lane.
4. Load other samples.
5. Run at 120 V until bromophenol blue migrates 2-3 cm.

Expected product: 448 bp.

1/18 12:00-6:00 pm:

PCR Product Purification

1. Discard the negative control tube.
2. Label the tubes you will need.
3. The remaining 45 μL of the rest of the PCR products need to be purified before performing a restriction digest.
4. Use the DNA Clean & Concentrator-5 Kit (Zymo Research, catalog D4014; Genesee Scientific, catalog 11-303C)
5. In a 1.5 mL microcentrifuge tube, add 5 volumes of DNA Binding Buffer to each volume of DNA sample.
   • I.e., to the 45 μL left of the PCR reaction, add 225 μL of DNA Binding Buffer.
   • Mix briefly by vortexing/flash spin.
6. Transfer mixture to a Zymo-Spin Column in a Collection Tube.
7. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
8. Discard the flow-through and replace the column in the collection tube.
9. Add 200 μL of DNA Wash Buffer to the column.
10. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
11. Discard the flow-through and replace the column in the collection tube.
12. Repeat the wash step by adding 200 μL of DNA Wash Buffer to the column.
13. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
14. Discard the flow-through and replace the column in the collection tube.
15. Spin the column again at 10,000 X g in the microcentrifuge to remove excess wash buffer. Make sure the column does not come in contact with the flow-through when removing it from the microcentrifuge and collecting tube.
16. Transfer the column to a clean 1.5 mL microcentrifuge tube with the cap cut off.
17. Elute the DNA by adding 20 μL of DNA Elution Buffer directly to the column matrix
18. Incubate at room temperature for 1 minute.
19. Spin for 30 seconds at 10,000 X g in the microcentrifuge.
20. Check and record volume of eluate as you transfer it to a 1.5 mL tube with lid. Store at -20°C or on ice if going on to the next step immediately.

• • Hela=20μL
  • KD=20μL
  • NM=μL
  • OO=μL

Restriction Digest with BsmFI

• Digest 0.4 μg of lambda DNA as a positive control. Bring 4 μL of 0.1 μg/mL lambda DNA to 10 μL by adding 6 μL sterile MilliQ water.
• Transfer 10 μL of purified PCR product to fresh 0.5 mL tube. (The remaining ~10 μL will be reserved as an uncut sample to run on the gel as a comparison).
• Make restriction digest master mix on ice with the restriction enzyme BsmFI (New England Biolabs cat# R0572S, isoschizomer with FaqI). Multiply the recipe by the number of tubes +0.5 to account for pipetting errors.
• Add the reagents in the order of water, then buffer. Mix and flash spin.
• Add the BsmFI. Mix and flash spin.

                               X1
DNA                          (10 μL)
10X rCutSmart Buffer           2 μL
sterile MilliQ water           7 μL
BsmFI enzyme (2 units/μL)      1 μL
Total                         20 μL

• Add 10 μL master mix to each tube, mix/flash spin.
• Incubate 60 minutes in thermocycler set at 65°C, followed by 10 minutes at 80°C to inactivate the enzyme. Program called BSMFI on small thermocycler.
  • Add 4 μL of 6X loading dye to the digested samples.
  • Add 2 μL of 6X loading dye to uncut DNA samples so that they can also be loaded on the gel.

Agarose Gel to Detect Restriction Fragments

• Pour 1.5% agarose gel with 1X TAE Buffer and final concentration of 0.5 μg/mL ethidium bromide (100 mL total volume of gel for large gel box).
• Load 10 μL of Quick-Load Purple 100 bp DNA Ladder (NEB cat# N0551S) to left-most lane.
• Load entirety of the restriction digest and uncut DNA samples on gel. Load reserved uncut PCR products to the left of the appropriate cut sample to facilitate easy comparison.
• Load the lambda DNA control digest in the right-most lane.
• Run gel at 120 volts.
  • CC genotype: two fragments of 351 and 97 bp
  • TT genotype: three fragments of 253, 98/97 bp
  • CT genotype: four fragments of 351, 253, and 98/97 bp

Week 3

1/24 12:00-6:00 pm:

Week 4

1/31 12:00-4:00 pm:

2/1 12:00-4:00 pm:

Week 5

2/7 12:00-2:00 pm:

2/8 12:00-6:00 pm:

Week 6

2/14 12:00-6:00 pm:

2/15 12:30-4:30 pm:

Week 7

2/21 12:15-4:15 pm:

Ran PCR Gel of Hela, 119, 167, 440, 918.

Hela and samples 119 and 440 were either degraded or contaminated so I did a new PCR of these samples & ran the resulting 1% gel.

2/22 12:30-4:30 pm:

Purification of PCR Products and Restriction Digest of Hela, 119, 167, 440, 918