Difference between revisions of "Template:Heidelberg/notebook/cf/week26"

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°

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

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

 

 

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.

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.

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.

 

 

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.