931 pET28b-PAK16D ligase

--Paul.xu (talk) 15:29, 21 December 2017 (EST)

12/27/2017

938 digestion with Nde1-Xho1, Fastdigest Buffer

1	2	3	4	5	6	7	8,9	10,11	13
938 PNGase_F	939 Tte-ExoIII	942 VVN	3122 CRM197	931, 1, PAK16D ligase	931, 2, PAK16D ligase	1855  yPolyA-Pol	1540	2046	2046
Nde1-Xho1	Nde1-Xho1	Nco1-Xho1	Nde1-Xho1	Nde1-HindIII	HindIII-Xho1	Nde1-Xho1	Nde1-Xho1	Nde1-Xho1	Nco1-Xho1
pJet1.2	pJet1.2	pJet1.2	pJet1.2	pJet1.2	pJet1.2	pEC2-	pET30b	pET28b	pET28b
1145	780	716	1798	1174	986	1713	there is no Xho1 site	1377	~1377

Date:4/5/2018

Subclone of 931

Vector:1987pT7A3

Insert:

F1:1174 bp

F2:986 bp

F1:Nde1+HindIII

F2:HindIII+Xho1

Date:5/29/2018

Subclone of 931

Vector is 1987pT7A3

Miniprep digestion of 984_931

#	Lane	Enzymes	Size	Note
984	2-5	Xba1+Nhe1	957bp	#6,7,,8 seems good
931	1,6-8	HindIII+Xho1	986bp	#5 seems good

Digest with HindIII +Xho1 and Nde1+ HindIII, Nde1 + Xho1

using parental vector as control. Need to have marker

not good

Date:7/10/2018

931-1 subclone

931 subclone

Miniprep digestion of 931-1

931 miniprep digestion 1

931-1 miniprep digestion

Nde1+HindIII

931subclone

TE as EB buffer

multiple bands in gel, not good, can not cut out the vector.

Date:8/17/2018

931-1  miniprep

vector:pET28b

insert:931-1 in 1727 vector

931 Subclone

vector is 931-1 in pET28b

HindIII+Xho1

Miniprep digestion of 931

931,#4  digestion

Lane	enzyme	size
1	Nde1+HindIII	1174
2	HindIII+Xho1	986
3	HindIII+Xho1	986
4	Nde1+Xho1	2267

Junk fragment , from hindIII+Xho1

Date:9/20/2018

931 PCR

931 vector digestion

931 PCR Screening