Contents 1 Materials 2 Methods 2.1 Restriction digestion for 5*UAS plasmid with EcoRI (unit: ul) 2.2 Electrophoresis to verify if most of the digested plasmid has been linearized 2.3 Heat-inactivation for the digestion system & Cold shock to prevent self-ligation 2.4 Do fill-in with T4 DNA polymerase 2.5 Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer 2.6 Blunt end ligation with T4 DNA ligase [Design a concentration gradient] 2.7 Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer 2.8 Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations 3 Results 3.1 Digested groups 3.2 Undigested groups 3.3 Improvement

UAS Fill-in (The second time)

2014/8/9 {{{goal}}}

Materials

The same as the first Fill-in experiment.

Methods

Restriction digestion for 5*UAS plasmid with EcoRI (unit: ul)

	Total volume	10X Buffer	DNA	EcoRI	ddH2O
Poly A(168.8ng/ul)	20	2	2.5	1	14.5
G-PB5(293.6ng/ul)	20	2	2	1	15

Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am (Incubate overnight)

Electrophoresis to verify if most of the digested plasmid has been linearized

Loading system：(unit: ul)

Total volume/well	DNA	Dye	TAE
12	0.5	2	9.5

Running conditions: 130V,   30min

Results of electrophoresis:

From the picture, we can see that, the position of the band of digested plasmid is apparently lying behind that of the original plasmid which hasn’t been digested by EcoRI, indicating that most of the plasmid in our digestion system has become linearized.

Heat-inactivation for the digestion system & Cold shock to prevent self-ligation

1. Heat-inactivation 75°C，10min

2. Chill on ice, 15min

Do fill-in with T4 DNA polymerase

1. Discard 0.5ul DNA from the former inactivated digestion system, forming the final 19ul system.

Till now, the concentration of DNA for:

Poly A: 21.1ng/ul

G-PB5: 29.36ng/ul

2. Fill-insystem：(unit: ul)

	Total volume	DNA	T4 DNA polymerase(3U/ul)	dNTP (10mM)	ddH2O
Poly A(21.1ng/ul)	20	19	0.2 (theoretical volume:0.13ul)	0.2	0.6
G-PB5(29.36ng/ul)	20	19	0.2 (theoretical volume:0.186ul)	0.2	0.6

[According to the manual provided by NEB：The NEBuffer 2.1 for EcoRI is also suitable for T4 DNA polymerase and, 1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]

3. Protocol:

a. incubate the fill-in system at 12°C，15min

b. add 2ul EDTA(100mM) to 18ul reaction system to stop the polyreaction.

c. put in 65°C，20min to inactivate T4 DNA polymerase thoroughly.

4. Accident：

To begin with, due to our carelessness, we added Taq DNA polymerase instead of T4 DNA polymerase to the 19ul system. But, thinking that it might not cause any serious consequence, we then just added T4 DNA polymerase into the system with the same volume.

Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer

DNA extraction:

Performed as the protocol provided by TIANgel Purification Kit, except that we eluted the DNA sample twice at the last step (as we always done before).

Concentration of the purified DNA fragments:

Poly A: 19.3ng/ul

G-PB5: 18.8ng/ul

Blunt end ligation with T4 DNA ligase [Design a concentration gradient]

1) Ligation system：（unit: ul）

	Total volume	DNA	10X T4 DNA ligase buffer	T4 ligase	EcoRI (to reduce those sticky ends ligation)	ddH2O
(40ng rank) 1.2 tube	21	2	2	1	1	15
(100ng rank) 3,4 tube	21	6	2	2	1	10
(200ng rank) 5,6 tube	21	12	2	2	1	4

2) Protocol：

a. incubate the ligation system at 16°C，15h [8.9 1:30pm ~ 8.10 4:30am ]

b. store at 4°C [8.10 4:30am ~10:00am]

c. Heat inactivation, 65°C, 10min

d. chill on ice

Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer

DNA extraction:

Performed as the protocol… but, we forgot to change the collecting tubes into EP tubes before adding EB solution…So, the most of the DNA was eluted into collecting tubes which might also contain many impurities(like ethanol, proteins…).

 

Anyway, we still transferred DNA in the collecting tubes into new EP tubes, and then detect the concentration of them.

	Concentration (ng/ul)	260/280	260/230
G-PB5 1,2	24.6	6.83	0.07
G-PB5 3,4	17.5	7.43	0.04
G-PB5 5,6	31.1	5.42	0.06
Poly A 1,2	21.4	5.07	0.06
Poly A 3,4	23.5	6.31	0.05
Poly A 5,6	25.8	5.44	0.06

Although everything seems normal, but we still decided not to use them.

 

To save our 2 day’s endeavor, we then put CA2 tubes into new EP tubes, and add 20ul EB solution, wishing to elute some DNA that haven’t been washed off for the first time. Fortunately, after two-time elution, we finally got relatively pure DNA samples.

	Concentration (ng/ul)	260/280	260/230
G-PB5 1,2	29.8	8.05	0.09
G-PB5 3,4	22.1	9.53	0.07
G-PB5 5,6	29.7	7.01	0.09
Poly A 1,2	22.9	11.53	0.07
Poly A 3,4	22.2	8.24	0.07
Poly A 5,6	19.9	11.63	0.06

  One abnormal phenomenon is that, in our ligation system, the total amount of DNA is 200ng or so at most. But from our extraction results, we can see that the quantity of DNA in each tube reaches 400ng around. So, where did these extra DNA come from? Or, is it just caused by the inaccurate measurement of Nanodrop?

Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations

1) Digestion system [unit: ul]

Total volume	DNA	EcoRI	10X NEBuffer
10	8.5	0.5	1

2) Reserve the rest 10ul DNA as backups

3) incubate the digestion system 37°C， 4h [12:35am ~ 5:00am]

9. Transformation

Performed as the usual protocol

1. Add 5ul DNA into 50ul competent cell. [Save the remaining 5ul DNA as backups]

2. We also transformed bacteria with those DNA that haven’t been digested by EcoRI as well. To make sure, if there were no colonies growing out, the reason shouldn’t lie in the ligation step.

3. Recover step: 200rpm, 45min, 18:44pm~19:30pm

4. Centrifuge before distributing on agar plate: 4000rpm, 5min

5. Incubate, 37°C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]

Results

Digested groups

Except G1,2 group with digested plasmid have colonies grown out, all the other digested groups have no colonies grown on. However, the colonies that grown on the G1,2 digested plates looked extremely abnormal. There are too many colonies growing on the plate that we nearly cannot distinguish any monocolones. Such strange result implied the failure of our second-time Fill-in.

Undigested groups

Some groups have colonies grown out on their undigested plates which can roughly indicate that our ligation was operative.

 

Improvement

1) Increase the quantity of DNA in each system

2) Heat inactivation before adding T4 DNA polymerase to avoid interplays.

3) Perform restriction digestion directly after ligation step without DNA extraction.

4) If we failed again for the third time, we plan to apply new method to eliminate the EcoRI site on UAS sequence. Such as, site-specific mutagenesis, or isocaudarner ligation.