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week number 26

▼2015-06-22 Miniprep and cryostock of culture 10 in colony PCR

Description:

 

Procedures:

 

Qiaprep Spin Miniprep Kit:

 

Description:

 

    Steps:

  1. Prepare o/n culture
  2. Perform mini prep according to manufacturers protocol

 

E.coli glycerol stocks:

 

Description:

 

    Steps:

  1. Grow up an overnight culture of strains of interest
  2. Transfer 500µl into a safe lock reaction tube
  3. Add 500µl of 40% sterile glycerol solution
  4. Freeze slowly at -80°C

 

Results:

 

Miniprep: 451,5ng/µl

2 stocks were frozen at -80°

▼2015-06-22 E.coli Trafo with Plasmids form the iGEM registry

To build the protein construct the mTagBFP BBa_K592100 from the iGEM registry were used.

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Do not remove the foil cover.

2. Pipette 10µl of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.

3. Thawing 50µl chemical competent E. coli on ice.

4. Add to 50µl competent cells:

1µl DNA from the registry

10µl KCM 5x

39µl H2O

5. Incubate on ice for 30 minutes

6. Heat shock at 42°C for 2 minutes

7. Incubate on ice for 2 minutes

8. Add 900 µl of LB or 2x YT Medium

9. Incubate on 37°C for 60min

10. Centrifuge 5min at 1000g

11. Take 900µl of supernatant and throw away

12. Resuspend pellet in remaining media

13. Plate out on agar with the appropriate antibiotic and grow overnight at 37°C

 

▼2015-06-23 Insert of MCS + promotor in cutted pSB1C3

Description:

 

Procedures:

 

Ligation:

 

Description:

 

        Materials and chemicals:

 

        2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature

        1 parts Vector DNA (mol not l)

        3 part Insert DNA (mol not l)

        20 µl Nuclease free water

        1 µl T4 DNA Ligase

 

    Steps:

 

Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.

 

        Gently mix the reaction by pipetting up and down and microfuge briefly.

 

        For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

 

For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.

 

        Heat inactivate at 80°C for 10 minutes

 

        Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.

 

 

Notes:

 

        Used fragments/chemicals:

 

        1 µl cutted pSB1C3

        0,1 µl MCS

        0,1 µl pCat

        2 µl T4 Buffer

        1 µl T4 Ligase

        Filled up to 20 µl with water

 

        Incubated for 2 hours at room temperature

 

Results:

 

Plated transformated bacteria (with ligation product) did not grew on a LB-Agar with CM

▼2015-06-23 PCR: Site-change of ribozyme constructs

Description:

 

Aim: Get every fragment to the same cloning standart.

BamHI -----------------------------/ BmtI

 

Procedures:

 

23.06.2015:

 

Description

 

        PCR mix:

 

        Polymerase Mastermix 2x: 25 µl

        Primer:        fwd: 1 µl

                               rev 1 µl

        Template-DNA 1 µl

        ddH20 22 µl

 

Number

Fragment Name

T Anneal

Fwd

Rev

1

CFTR 1 T

58°C

DH13

DH14

2

CFTR 1 A

58°C

DH13

DH14

3

CFTR 1 C

58°C

DH13

DH14

4

CFTR 2 A

61°C

DH13

DH15

5

CFTR 2 C

61°C

DH13

DH15

6

GFP 1

61°C

DH16

DH17

 

 

Results:

 

1: 1 band + smear: PCR settings were not right --> many sideproducts were synthesized

2: 2 bands + smear: As above, lower band is our desired product

3: 2 bands: Higher product yield than 1 and 2, higher purity than 1 and 2

4: One small band: Low yield and higher purity than 3

5: One small band: Lower yield than 4

6: Smear: No visible product band

 

The PCR settings were wrong for our target DNA.

 

24.06.2015:

 

Description:

 

        PCR-Mix:

        25 µl Polymerase Master Mix 2x

        1 µl Primer fwd

        1 µl Primer rev

        1 µl Template-DNA

        22 µl ddH2O

 

Number

Template

T Anneal

Fwd

Rev

C(ng/µl)

Comment

1

CFTR 1 T

60°C

DH13

DH14

24

Number 1 + 1a in one tube

2

CFTR 1 A

60°C

DH13

DH14

30,5

Number 2 + 2a in one tube

3

CFTR 1 C

60°C

DH13

DH14

25

Number 3 + 3a in one tube

4

CFTR 2 A

60°C

DH13

DH15

8,1

 

5

CFTR 2 C

60°C

DH13

DH15

8,2

 

6

GFP 1

60°C

DH16

Dh17

-0,6

 

1a

CFTR 1 T

57°C

DH13

DH14

24

 

2a

CFTR 1 A

57°C

DH13

DH14

30,5

 

3a

CFTR 1 C

57°C

DH13

DH14

25

 

4a

CFTR 2 A

57°C

DH13

DH15

7,2

 

5a

CFTR 2 C

57°C

DH13

DH15

 

 

6a

GFP 1

57°C

DH16

DH17

 

 

 

 

Results:

1/2 = 1/1a

3/4 = 2/2a

5/6 = 3/3a

7/8 = 4/4a

9/10 = 5/5a

10/11 = 6/6a

 

 

1/2: 60°C is the optimal temperature for our PCR

3/4: 60°C and 57°C yield equal amounts of DNA

5/6: 60°C and 57°C yield equal amounts of DNA

7/8: 60°C yields no DNA, 57°C yields less DNA than 1/2

9/10: 60°C yields no DNA, 57°C yields less DNA than 7/8

11/12: Smear, no band at 60°C and 57°C

 

The Annealing Temperatures for most products are help to get a high yield. Just for 9/10 and 11/12 the yields are not optimal.

 

25.06.2015:

 

Description:

 

PCR-Mix:

 

Like in the former protocols

 

Number

Template

T Anneal

Fwd

Rev

5.1

CFTR 2 C

55°C

DH13

DH15

5.2

CFTR 2 C

53°C

DH13

DH15

6.1

GFP 1

55°C

DH16

DH17

6.2

GFP 1

53°C

DH16

DH17

 

 

▼2015-06-23 Digest of pSB1C3 BBa_J04450

Description:

 

Procedure:

 

20 µl test-digest:

 

Description

 

    Steps:

 

  1. Set up reaction according to protocol:

                ddH2O for a final volume of 20 µl

                2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)

                0.5 µl of selected Enzyme(s)

                ca. 1 µl of mini prep DNA (Range 200-1000 ng)

 

  1. Incubate at 37°C for 60'

 

  1. Load on gel (add loading dye first)

 

Notes:

 

        Selected Enzymes:

        EcoRI/SpeI

 

        Buffer:

        Cutsmart

 

        Heat inactivation at 60°C

 

        Everything was given on the gel

 

        A fragment with about 2 kb was cut out

 

        Gel elution: Result: 50 ng/µl DNA

 

Results:

 

50 ng/µl DNA

 

5:

Title: Yeast Transformation with p413-GPD and CFTR construct 2

Author: Hendrik

Date: 23.06.2015

 

Description:

 

Procedures:

 

 

Yeast transformation:

 

 

Description:

 

                10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR                       product

                2 µl of plasmid DNA per 10 µl of cells

                6 equivalents of PEG

                1/9 equivalents of DMSO

                100 - 200 µl of liquid medium

   

                Steps:

 

  1. Give the plasmid DNA into a Eppi. Add the competent cells.
  2. Mix, then add the PEG.
  3. Incubate for 30 mins at room temperature while mixing
  4. Add the DMSO
  5. Place the yeast in a 42°C water bath for 5-20 minutes
  6. Centrifuge cells for 2-3 minutes at 2000 rpm
  7. Discard the supernatant and resuspend the yeast in the liqiud YPD medium

 

Notes:

 

        10 µl of Yeast and 100 µl of SD-His medium was taken.

        2 transformations were made

        After transformation the yeast was plated.

 

Results:

 

Some yeast grew on the first plate, but not on the second plate (I took some of the original biofilm and made a fractionated plating). Also the biofilm on the first plate exhibited no further growth. Maybe the wrong yeast medium was token (The writing on the flasks didn't survive the autoclave). Another plating was been made to see if this is right. The yeast on the SD-His and SD-Leu plate exhibited no growth. The yeast in the SD-His/Leu plate grew spotlike and maybe forms colonies.

▼2015-06-23 Yeast Transformation with p413-GPD and CFTR construct 2

Description:

 

Procedures:

 

 

Yeast transformation:

 

 

Description:

 

                10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR                       product

                2 µl of plasmid DNA per 10 µl of cells

                6 equivalents of PEG

                1/9 equivalents of DMSO

                100 - 200 µl of liquid medium

   

                Steps:

 

  1. Give the plasmid DNA into a Eppi. Add the competent cells.
  2. Mix, then add the PEG.
  3. Incubate for 30 mins at room temperature while mixing
  4. Add the DMSO
  5. Place the yeast in a 42°C water bath for 5-20 minutes
  6. Centrifuge cells for 2-3 minutes at 2000 rpm
  7. Discard the supernatant and resuspend the yeast in the liqiud YPD medium

 

Notes:

 

        10 µl of Yeast and 100 µl of SD-His medium was taken.

        2 transformations were made

        After transformation the yeast was plated.

 

Results:

 

Some yeast grew on the first plate, but not on the second plate (I took some of the original biofilm and made a fractionated plating). Also the biofilm on the first plate exhibited no further growth. Maybe the wrong yeast medium was token (The writing on the flasks didn't survive the autoclave). Another plating was been made to see if this is right. The yeast on the SD-His and SD-Leu plate exhibited no growth. The yeast in the SD-His/Leu plate grew spotlike and maybe forms colonies.

▼2015-06-24 QIAprep Spin Miniprep Kit

Miniprep with mTagBFP BBa-K592100 and the pUB23-S-ßGal plasmid was performed.

1. Prepare o/n culture

2. Perform mini prep according to manufacturer’s protocol

Durchführung

▼2015-06-25 PCR of the Fragments E, B-Hammerhead Ribozyme (Hammerhead ribozyme: HHR), EGS-Hammerhead Ribozyme and M1

Aim: Amplification of the DNA-primers for the in vitro-transcription

 

Procedure:

  1. Make a 1:10 thinner of each primer by using each 9 µL water and 1 µL primer

 

 

Fragment primer

Forward Primer

Reverse Primer

Lenght with out T7 promotor [bp]

Fragment E

FS01, FS02

FS 13

FS 14

66

Fragment B

FS04, FS05

FS 13

FS 15

54

Fragment EGS

FS10, FS

FS 13

FS 16

65

Fragment M1

genomic DNA

FS 08

FS 09

  

 

  1. Fill up the following ingredients in a PCR-tube and mix them well:

    1. 1 µL of 1 µM fragment  

    2. 19 µL of water

    3. 2,5 µL of 10 µM reverse primer

    4. 2,5 µL of 10 µM foward primer

    5. 25 µL of 2x PCR Mastermix Phuion Flash

  1. Put the samples into a PCR-cycler (cycle is discribed below):

    1. 98 °C for 2:30 min (denaturation)

    2. 98 °C for 0:15 min

    3. 55 °C for 0:15 min (annealing)

    4. 72 °C for 0:20 min (polymerisation)

    5. Go to step 2 and repeat for 39x

    6. 72 °C for 2:00 min

    7. 4 °C for cool down

Time total: 1 h 20 min 54 sec

  1. Analyze the PCR-products by using an 1 % agarose gel with ethidumbromid:

    1. 1 % agarose

    2. 1 x TAE

    3. 20 µL/L Ethidumbromid for gel

  2. Put 5 µL of each PCR-product which was mixed with 2 µL Loading Dye in a pocket

  3. Fill 5 µL of a 2 log Ladder in the last pocket

  4. Let the gel run for 30 min at 120 V.

  5. Analyze the gel by using UV-light.

 

Conclusion:

  • The PCR has worked, therefore the fragments are amplified

  • Furthermore the primers (forward and reverse) were designed right since there is no visible ladder for them

▼2015-06-25 E. coli glycerol stocks

Glycerol stocks from E.coli containing mTag BFP BBa_K592100 or pUB23-S-ßGal were made.

1. Grow up an overnight culture of strains of interest

2. Transfer 500µl into a safe lock reaction tube

3. Add 500µl of 40% sterile glycerol solution

4. Freeze slowly at -80°C

Durchführung

▼2015-06-25 Fragment amplification for protein construct (PCR)

To obtain the required Gibson Fragments (Backbone (p413-GPD), mTagBFP, Ubiquitin, sfGFP) PCRs were performed.

 

reaction mixture:

12.5µl Q5 High-Fidelity 2x Master Mix

0.5µl DNA

1.25µl fwd primer 10µM

1.25µl rev primer 10µM

9.5µl H2O

 

Program:

95°C 2min

95°C 30sec

63-66°C 30sec (mTagBFP: 63°C; Ub: 65°C; sfGFP: 64°C; p415-GDP: 66°C)

72°C 30sec

72°C 5min

35 cycles

 

gel:

TAE buffer

1% w/v agarose

 

loading samples:

5µl Ladder

25µl PCR sample

5µl Loading buffer

▼2015-06-26 Precipitation of the PCR-DNA

Aim: Cleaning the DNA as a prepartional step for the in vitro transcripton

 

Procedure:

  1. Mix each PRC-Product with 0.1x V of a 3 M NaAc and 2.5x V of absolute ethanol

  2. Vortex the mixture well

  3. Put the samples in the freezer for overnight

  4. On the next day, spin the DNA-Ethanol mixture at 4 °C with 13000 rpm for 30 min in a centrifuge

  5. Remove the ethanol from the pellet and rehydrate the pellet with 1 ml of 70 % absolute ethanol.

  6. Vortex again.

  7. Repeat the steps 4 to 6 for one more time and for the last cylce just 4

  8. Remove the ethanol from the pellet and rehydrate the pellet with 100 µL millipore water.

  9. Vortex the sample again.

▼2015-06-27 Test if transformed cells have the MCS + pcat insert

Description:

 

Procedures were done on 5 ml overnight E.coli culture with estimated MCS+pCat insert in pSB1C3

Five colonies were picked from the original plate and were given in 5 snapcaps with 5 ml LB medium each.

The 5 cultures are named 1-5

 

Procedures:

 

QIAprep Spin Miniprep Kit:

 

Description:

 

    Steps:

 

  1. Prepare o/n culture

 

  1. Perform mini prep according to manufacturers protocol

 

Notes:

 

After the miniprep 2 ml of overnight culture were transferred to 100 ml of fresh LB medium with 1:1000 Chloramphenicol

 

Results:

 

Nanodrop results:

1: c = 9,5 ng/µl

2: c = 7,7 ng/µl

3: c = 81,5 ng/µl

4: c = 48,5 ng/µl

5: c = 64 ng/µl

 

A test digestion will be made with 3-5. The concentrations for 1-2 are too low.

 

20 µl test-digest:

 

Description

 

    Steps:

 

  1. Set up reaction according to protocol:

                ddH2O for a final volume of 20 µl

                2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)

                0.5 µl of selected Enzyme(s)

                ca. 1 µl of mini prep DNA (Range 200-1000 ng)

 

  1. Incubate at 37°C for 60'

 

  1. Load on gel (add loading dye first)

 

Notes:

 

                Digest with EcoRI and SpeI

                Heat inactivation at 65°C for 20 minutes

                The gel picture should have one band when the plasmid have no insert (the natural plasmid   has no SpeI cutting site) and two bands when the insert is in the plasmid (the MCS has one              cutting site for SpeI).

 

Results:

 

The agarose gel shows, that none of our colonies has got the MCS + pCat fragment.

 

 

▼2015-06-27 Transformation test for pSB1C3 + MCS and pcat

Description:

 

Procedures were done on 5 ml overnight E.coli culture with estimated MCS+pCat insert in pSB1C3

Five colonies were picked from the original plate and were given in 5 snapcaps with 5 ml LB medium each.

The 5 cultures are named 1-5

 

Procedures:

 

QIAprep Spin Miniprep Kit:

 

Description:

 

    Steps:

 

  1. Prepare o/n culture

 

  1. Perform mini prep according to manufacturers protocol

 

Notes:

 

After the miniprep 2 ml of overnight culture were transferred to 100 ml of fresh LB medium with 1:1000 Chloramphenicol

 

Results:

 

Nanodrop results:

1: c = 9,5 ng/µl

2: c = 7,7 ng/µl

3: c = 81,5 ng/µl

4: c = 48,5 ng/µl

5: c = 64 ng/µl

 

A test digestion will be made with 3-5. The concentrations for 1-2 are too low.

 

20 µl test-digest:

 

Description

 

    Steps:

 

  1. Set up reaction according to protocol:

                ddH2O for a final volume of 20 µl

                2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)

                0.5 µl of selected Enzyme(s)

                ca. 1 µl of mini prep DNA (Range 200-1000 ng)

 

  1. Incubate at 37°C for 60'

 

  1. Load on gel (add loading dye first)

 

Notes:

 

                Digest with EcoRI and SpeI

                Heat inactivation at 65°C for 20 minutes

                The gel picture should have one band when the plasmid have no insert (the natural plasmid   has no SpeI cutting site) and two bands when the insert is in the plasmid (the MCS has one              cutting site for SpeI).

 

Results:

 

The agarose gel shows, that none of our colonies has got the MCS + pCat fragment.

▼2015-06-27 Fragment amplification for protein construct (PCR) (continued)

Results

PCRs for Gibson fragments mTagBFP, Ubiquitin and sfGFP worked because a bands are visible with the expected size. No p415-GPD product was visible. Therefore the p415-GPD PCR need to be optimised.

Gel Extraction

mTagBFP, Ubiquitin and sfGFP fragments were separated in 1% agaros.

Gel extraction was performed according to QIAGEN QIAquick Gel Extraction Protocol. (Elution in 30µl EB)

▼2015-06-27 Phusion PCR with DMSO and GC Buffer

To amplify the p415-GPD plasmid with Gibson overhangs an optimized gradient PCR program was used.

 

reaction mixture:

total:10µl

5.0µl Phusion 2x Master Mix

0.2µl DNA

0.5µl xxJD007xx 10µM

0.5µl xxJD008xx10µM

0.5µl DMSO

 

1.3 H2O

 

programm:

(PHGRAD)

95°C 2:00min

95°C 0:30min

58°C-70°C 0:30min

72°C 3:00min

72°C 5:00

35 cycles

 

gel:

TAE buffer

1% w/v agarose

 

loading samples:

5µl PCR sample

2µl Loading buffer

 

Results

no product. PCR need to be repeated.

 

 

▼2015-06-27 Phusion PCR with DMSO and GC Buffer

To amplify the p415-GPD plasmid with Gibson overhangs an optimized gradient PCR program was used.

 

reaction mixture:

total:10µl

5.0µl Phusion 2x Master Mix

0.2µl DNA

0.5µl xxJD007xx 10µM

0.5µl xxJD008xx10µM

0.5µl DMSO

 

1.3 H2O

 

programm:

(PHGRAD)

95°C 2:00min

95°C 0:30min

58°C-70°C 0:30min

72°C 3:00min

72°C 5:00

35 cycles

 

gel:

TAE buffer

1% w/v agarose

 

loading samples:

5µl PCR sample

2µl Loading buffer

 

Results

no product. PCR need to be repeated.

 

▼2015-06-27 p415-GPD test digest

To check if the right plasmid is in the tube a KpnI digest was performed.

Set up reaction according to protocol:

  • ddH2O for a final volume of 20 µl
  • 2 µl of 10x 1.1 Reaction Buffer
  • 0.5 µl of KpnI
  • 1 µl of DNA

Incubate at 37°C for 60'

Load on gel (add loading dye first)

 

Results

The gel shows the expected bands. The p415 plasmid is in the tube.

 

 

▼2015-06-27 in vitro-transcription of the fragments E, B-HHR, EGS-HHR and M1 out of the DNA that was generated out of the PCR form the 25th June 2015

Aim: transcribe the PCR-samples into RNA to have the single components for the construct

Procedure for 100 µL transcription:

  1. Mix together

    1. 4 µL of 100 mM each NTP (ATP, GTP, CTP and UTP)

    2. 10 µL of 10 x transcription buffer

    3. 1 µL of 1 M DTT

    4. 5 % of DMSO

    5. 5 µL of T7 ( 2 mg/mL)

    6. 10 µL of the DNA-template

    7. and 53 µL millipore water

  2. Use a pipett to mix it up and down (do not vortex)

  3. Put the samples into a heatblock at 37 °C for 3 hours

  4. Add  to the mixture 5 µL of T7 polymerase and 2 µL of 1 M MgCl2 addtionally after 2 hours

  5. Add 4 µL DNase I to the mixture and let it incubate at 37 °C for 20 min.

▼2015-06-27 Quantative Gel for RNA-Extraction

Aim: Purifying the RNA that was transcribed in the previous in vitro assay

Procedure:

  1. Make a 8 M urea polyacrylamid gel with  Roth© mastermix, 400 µL APS and 40 µL TEMED.

  2. Mix the sample with 2x Loading Dye to get a 1x Loading Dye concentration

  3. Clean the pockets with TBA buffer

  4. Load the entire in vitro-Transcript with Loading Dye in a pocket

  5. Fill TBA buffer in all reservoirs of the gel system

  6. Run the gel at 300 V for 3 hours

  7. Analyze the gel through UV-Shadowing by using a TLC-plate underneath your gel