DATE:8.20

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Mix gently and incubate at 22 degree Celsius for an hour.

The number of the product: YBL8201002 and YBL8201003

Then we do transformation.

**Recorder: Chenyang Li ** PCR of Sup35

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	sup35
nuclease-free water(μL)	30.5
5×PrimeStar Buffer(μL)	10
dNTP(μL)	4
template(μL)	4
PrimeStar	0.5
Primer1	0.5
Primer2	0.5

50 μL in total.

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95	7 min
Duration	95	10s
Anneal	57	15s
Extend	72	50s
Post-Extend	72	10 min
Final	4	--

35 cycles(Duration ~ Extend)

Purification for the product of the PCR of Sup35 Recorder: Chenyang Li

results

Number	Sup35P8201001	Sup35P8201002	Sup35P8201003	Sup35P8201004
A260/A280	1.87	1.88	1.86	1.87
A260/A230	2.29	2.29	1.91	2.13
concentration(ng/μL)	170.2	302.3	334.6	301.0

**Recorder：Chenyang Li ** Double Digestion of YeWGAP,BD

Procedure:

number	1	2	3	4
sample	Sup35P8201002	Sup35P8201002	Sup35P8201004	Sup35P8201004
nuclease-free water(μL)	4	7.8	4	7.8
10* Fast Digest Buffer(μL)	2	2	2	2
plasmid(μL)	10	7	10	7
EcoRI(μL)	3	2.4	3	2.4
Bcul(μL)	2.4	0.8	2.4	0.8

20 μL in total. Mix 1,2,3,4gently and incubate at 37 degree Celsius for 15 min.

result: (from left to right: Trans 2K plus 2(contain Gelred),1,2,3,4,PSB1C3+AD1,PSB1C3+AD2,PSB1C3+AD3,PSB1C3+AD4,)

DATE:8.22

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Mix gently and incubate at 22 degree Celsius for an hour.

The number of the product: YBL8201004. Then we do transformation.

PCR of sup35 **Recorder: Yinchenguang Lyu **

21μL ddH₂O 25μL 2×HiFi PCR Master 2μL template 1μL primer1 1μL primer2

stage	temperature	time
step 1	95	5 min
step 2	95	50 s
step 3	56	1 min
step 4	72	1 min 50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

DATE 8.23

Plasmid Extraction for ligased PSB1C3 and AD Recorder: Yinchenguang Lyu

results

sample	1	2	3	4
A260/A280	1.81	1.77	1.70	1.57
A260/A230	1.64	1.43	0.79	0.78
concentration(ng/μL)	175.9	204.4	216.3	346.1

Recorder：Yu Xie Colony picking of plasmid pUC57 containing sfGFP11

We did colony picking of plasmid pUC57 containing sfGFP11. After colony picking, we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Junjie Zeng & Yu Xie PCR of sup35

Procedure:

1.Prepare 8 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7,8
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95℃	10 min
Duration	95℃	1 min
Anneal	58℃	1 min
Extend	72℃	1 min 50 s
Post-Extend	72℃	10 min
Final	4℃	--

30 cycles(Duration ~ Extend)

Recorder: Yu Xie Double digestion of sfGFP11

Recorder: Yu Xie Double digestion of sup35