Heidelberg/notebook/cf/week37

week number 37

▼2015-09-07 Cloning of ribozyme 12-1 (Ribozyme without Insert) from pSB1C3 into p413-GPD

Procedures:

 

Digestion:

 

Description:

 

Materials and Chemicals:

4 µl of pSB1C3 with pcat-MCS and ribozyme 12, about 2 µg of DNA in total

2 µl Cutsmart Buffer

1 µl BamI-HF

1 µl SalI-HF

12 µl ddH2O

 

The digest was incubated for 1 hour at 37°C while mixing slowly

 

The lower band with cut out insert (ribozyme 12) was purificated by gel extraction

 

Ligation:

 

Description:

 

Materials and Chemicals:

1 µl of p413-Vector DNA

6 µl of ribozyme 12 insert (purificated by gel extraction)

1 µl of T4-Ligase

2 µl of T4-Ligase Buffer

10 µl ddH2O

 

 

The ligation was incubated for 30 minutes at room temperature (about 25°C)

 

KCM Transformation:

 

Description:

 

Steps:

 

  1. Take 50µl chemical competent E. coli from -80 freezer and thaw on ice

 

  1. Add (as master mix):

                2,5µl DNA

                10µl KCM 5x

                37,5µl H2O

 

  1. Incubate on ice for 30 minutes

 

  1. Heat shock at 42°C for 1 minute

 

  1. Incubate on ice for 2 minutes

 

  1. Add 900 µl of LB or 2x YT Medium

 

  1. Incubate on 37°C for 60min

 

  1. Centrifuge 5min at 1000g

 

  1. Take 900µl of supernatant and throw away

 

  1. Resuspend pellet in remaining media

 

  1. Plate out on agar with antibiotics (1:1 / 1:10)

 

Used DNA: Ligation of p413 and ribozyme 12-1

10 µl instead of 2,5 µl were used

 

▼2015-09-09 PCR of Type-1 Ribozymes for in vitro assay

 

Ribozyme CFTR1 A

[µl]

Ribozyme CFTR1 C

[µl]

Ribozyme CFTR1 T

[µl]

Primer Fwd

DH_82

5

DH_82

5

DH_82

5

Primer Rev

DH_33

5

DH_83

5

DH_83

5

Template

Ribozyme CFTR1 DE A

0,5

Ribozyme CFTR1 DE C

0,5

Ribozyme CFTR1 DE T

0,5

ddH2O

 

14,5

 

14,5

 

14,5

Q5 Polymerase

 

25

 

25

 

25

 

Conditions:

Step

Temperature [°C]

Time

Cycles

Initial denaturation

98

1:00

1

Denturation

98

0:10

35

Annealing

66

0:15

Extension

72

0:15

Final Extension

72

1:00

1

Hold

4

 

Cycles

 

After PCR DNA was purified by Qiagen PCR Purification Kit ans stored at -20°C.

 

▼2015-09-10 In vitro transcription of CFTR1-type ribozymes

 

For every reaction:

Stock solution

Final concentration

[µl]

ATP 100mM

4 mM

8

CTP 100mM

4 mM

8

UTP 100mM

4 mM

8

GTP 100mM

4 mM

8

DTT 1M

1 mM

2

DMSO

5%

10

10x Transcription buffer

1x

20

DNA

1 µg

20

ddH20

 

106

T7 RNA polymerase

 

3

 

 

  • Reaction was incubated for 3 h at 37 °C
  • After 1.5 h another 2 µL T7 RNA Polymerase were added
  • Addition of 2 µL DNase I and further incubation at 37 °C for 20 min
  • Reaction was heated up to 95° for 5 min, so that there is a coplete cleavage.

 

RNA purification by precipitation:

  • Samples was mixed with 200 µL of 2 x loading dye and purified over a 10 % PAGE
  • Bands were visualized by UV shadowing and suitable bands were excised
  • RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
  • Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
  • Spin sample at 16,000 g for 30 min, discard the supernatant
  • Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water