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Team:Georgia State/Notebook/august
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Restriction digest of Miniprep pGAPzαB
8/19/13
Page 138 Alisha’s notebook
Gel protocol
1. 1g of Agrose powder
2. 100ml of 1X TAE Buffer
3. Microwave for 2 minutes at 30 second intervals
4. Allow to cool down until cool to the touch
5. Add 10ul of gel red stain to gel
6. Allow the gel to settle for at least 45 minutes (the longer the better)
7. Once solidified load samples
Protocol
1. 50ul of Miniprep DNA
2. 10ul of 10x Buffer
3. 1ul of Xho1
4. 1ul of Xba1
5. 38ul of H2O 100ul of total volume
6. Incubation for 10 minutes at 37°C
7. Add 10ul of 3M NaAC
8. Add 250ul of chilled 100% ethanol 9. Incubate at -80°C for 20 minutes to allow for precipitation
10. Centrifuge for 15 minutes at 10,000xg at 4°C
11. Remove the supernatant
12. Wash the pellet with chilled 50ul of 70% ethanol then centrifuge at 10,000xg for 15minutes at 4°C
13. Remove supernatant and place into the air vac
14. Resuspend the pellet in 45ul of TE Buffer
15. Add 9ul of 6x loading dye
16. Load samples Lanes
1. Empty
2. Empty
3. 1kb GeneRuler ladder (2ul)
4. Empty
5. Digested miniprep pGAPzαB (54ul)
6. Empty
7. Bste II digest ladder (2ul)
8. Empty
9. Empty
Ran gel for 999minutes at 15V
A band was found between 3500 and 3000 kb
Gel exertion and extraction were performed
Gel Extraction
8/20/13
Page 140 Alisha’s Notebook
GeneJet Kit
Gel is 0.63g
Protocol
1. Excise gel slice containing the DNA fragment using a clean scalpel and placing it in a clean 1.5ml tube. Record the weight of the gel slice.
2. Add 1:1 volume of Biding Buffer to the gel slice.
3. Incubate the gel mixture at 50-60°C for 10 minutes or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to help the melting process. Ensure the gel is completely dissolved.
4. Transfer 800ul of the gel solution to the GeneJET purification column. Centrifuge for 1 minute. Discard the flow-through and place the column back into the same collection tube. If the volume is more than 800ul then do an additional spin.
5. Add 100ul of Binding Buffer to the column. Centrifuge for 1 minute. Discard the flow-through
6. Add 700ul of Wash Buffer to the column. Centrifuge for 1 minute. Discard the flow-through.
7. Centrifuge the empty column for an additional minute to completely remove residual wash buffer.
8. Transfer the GeneJet column into a clean 1.5ml tube. Add 50ul of Elution Buffer to the center of the column.
9. Store flow-through in -20°C in Merhawi’s box.
The minipreps from the week before had low DNA concentrations so we used gel purification to clean the samples.
We transformed E. coli with the RFP from Kit Plate 3, 8L and plated it. We also plated some of the pGAPZαA + Unknown RFP for comparison.
We found a stop codon in the middle of the cDNA of the Mambalgin I protein that we had designed. We removed the stop codon and change the cDNA so that it would fall in the reading frame of pGAPZαC instead of pGAPZαB, since we were closer to standardizing pGAPZαC. We sent the new sequence to IDT labs.
Week 14: 8/12 - 8/16
We ligated two samples of pGAPZαC + Linker with the new Mambalgin I with two different ligases. We used chemical transformation of samples, and let the samples recover in SOC broth overnight in the 37°C shaking incubator. Then plated the samples on selective media plates the next day.
The transformed E. coli cells with pGAPZαC + Linker + Mambalgin I grow so we performed minipreps on an overnight culture. The we digested the minipreps using Bgl II and Xba I, Eco RI and Pst I, and Avr II. We ran the samples in a 0.8% agarose gel at 70 V for 110 minutes.
We also digested pGAPZαC+ Linker using EcoRi and PstI. Then we gel isolated the sample to increase the purity of the sample.
Week 15: 8/19 - 8/23
We digested samples of pGAPZαC + Linker + Mambalgin I and pGAPZαC + Linker with the same enzymes to verify the insertion of the Mambalgin I cDNA. We performed double digestions using EcoRI and PSt I, Bgl II and Xbal, Bgl II and BamHI, and did a triple digestion using Bgl II, Xbal, and BamHI.
We also performed whole cell PCRs of E. coli with the pGAPZαC + Linker + Mambalgin I and ran all the samples on the same gel. We ran the samples on a .8% agarose gel at 30 V for 1010 minutes.
The colony PCRs had a large concentration of DNA around 200 bp that was unexpected so reran the colony PCRs but recieved the same strange ban around 200 bp.
Week 16: 8/26 - 8/30
We ran colony PCRs on the pGAPZαC + Linker + Mambalgin I using a different set of forward and reverse primers, but continued to have unexpected bands.
Since the digestions had verified the insertion of the Mambalgin I cDNA, we linearized minipreps with Avr II. We performed an ethanol precipitation on the samples, then isolated the cDNA by running it on a .8% agarose gel at 15V for 999 minutes. We then gel purified the sample.
Alisha/Merhawi, Group A
Restriction digest of Miniprep pGAPzαB
8/19/13
Page 138 Alisha’s notebook
Gel protocol
1. 1g of Agrose powder
2. 100ml of 1X TAE Buffer
3. Microwave for 2 minutes at 30 second intervals
4. Allow to cool down until cool to the touch
5. Add 10ul of gel red stain to gel
6. Allow the gel to settle for at least 45 minutes (the longer the better)
7. Once solidified load samples
Protocol
1. 50ul of Miniprep DNA
2. 10ul of 10x Buffer
3. 1ul of Xho1
4. 1ul of Xba1
5. 38ul of H2O 100ul of total volume
6. Incubation for 10 minutes at 37°C
7. Add 10ul of 3M NaAC
8. Add 250ul of chilled 100% ethanol 9. Incubate at -80°C for 20 minutes to allow for precipitation
10. Centrifuge for 15 minutes at 10,000xg at 4°C
11. Remove the supernatant
12. Wash the pellet with chilled 50ul of 70% ethanol then centrifuge at 10,000xg for 15minutes at 4°C
13. Remove supernatant and place into the air vac
14. Resuspend the pellet in 45ul of TE Buffer
15. Add 9ul of 6x loading dye
16. Load samples Lanes
1. Empty
2. Empty
3. 1kb GeneRuler ladder (2ul)
4. Empty
5. Digested miniprep pGAPzαB (54ul)
6. Empty
7. Bste II digest ladder (2ul)
8. Empty
9. Empty
Ran gel for 999minutes at 15V
A band was found between 3500 and 3000 kb
Gel exertion and extraction were performed
Gel Extraction
8/20/13
Page 140 Alisha’s Notebook
GeneJet Kit
Gel is 0.63g
Protocol
1. Excise gel slice containing the DNA fragment using a clean scalpel and placing it in a clean 1.5ml tube. Record the weight of the gel slice.
2. Add 1:1 volume of Biding Buffer to the gel slice.
3. Incubate the gel mixture at 50-60°C for 10 minutes or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to help the melting process. Ensure the gel is completely dissolved.
4. Transfer 800ul of the gel solution to the GeneJET purification column. Centrifuge for 1 minute. Discard the flow-through and place the column back into the same collection tube. If the volume is more than 800ul then do an additional spin.
5. Add 100ul of Binding Buffer to the column. Centrifuge for 1 minute. Discard the flow-through
6. Add 700ul of Wash Buffer to the column. Centrifuge for 1 minute. Discard the flow-through.
7. Centrifuge the empty column for an additional minute to completely remove residual wash buffer.
8. Transfer the GeneJet column into a clean 1.5ml tube. Add 50ul of Elution Buffer to the center of the column.
9. Store flow-through in -20°C in Merhawi’s box.
Kristin/Lydia, Group C
Week 13: 8/5 - 8/9The minipreps from the week before had low DNA concentrations so we used gel purification to clean the samples.
We transformed E. coli with the RFP from Kit Plate 3, 8L and plated it. We also plated some of the pGAPZαA + Unknown RFP for comparison.
We found a stop codon in the middle of the cDNA of the Mambalgin I protein that we had designed. We removed the stop codon and change the cDNA so that it would fall in the reading frame of pGAPZαC instead of pGAPZαB, since we were closer to standardizing pGAPZαC. We sent the new sequence to IDT labs.
Week 14: 8/12 - 8/16
We ligated two samples of pGAPZαC + Linker with the new Mambalgin I with two different ligases. We used chemical transformation of samples, and let the samples recover in SOC broth overnight in the 37°C shaking incubator. Then plated the samples on selective media plates the next day.
The transformed E. coli cells with pGAPZαC + Linker + Mambalgin I grow so we performed minipreps on an overnight culture. The we digested the minipreps using Bgl II and Xba I, Eco RI and Pst I, and Avr II. We ran the samples in a 0.8% agarose gel at 70 V for 110 minutes.
We also digested pGAPZαC+ Linker using EcoRi and PstI. Then we gel isolated the sample to increase the purity of the sample.
Week 15: 8/19 - 8/23
We digested samples of pGAPZαC + Linker + Mambalgin I and pGAPZαC + Linker with the same enzymes to verify the insertion of the Mambalgin I cDNA. We performed double digestions using EcoRI and PSt I, Bgl II and Xbal, Bgl II and BamHI, and did a triple digestion using Bgl II, Xbal, and BamHI.
We also performed whole cell PCRs of E. coli with the pGAPZαC + Linker + Mambalgin I and ran all the samples on the same gel. We ran the samples on a .8% agarose gel at 30 V for 1010 minutes.
The colony PCRs had a large concentration of DNA around 200 bp that was unexpected so reran the colony PCRs but recieved the same strange ban around 200 bp.
Week 16: 8/26 - 8/30
We ran colony PCRs on the pGAPZαC + Linker + Mambalgin I using a different set of forward and reverse primers, but continued to have unexpected bands.
Since the digestions had verified the insertion of the Mambalgin I cDNA, we linearized minipreps with Avr II. We performed an ethanol precipitation on the samples, then isolated the cDNA by running it on a .8% agarose gel at 15V for 999 minutes. We then gel purified the sample.
