Yijia LI 2 19 2018

Date:2/20/2018

#	Template	2X PCR Mix	Primers
3027	2492-1,2,3	Q 5	3020-Xba1-Nhe1-F + 3027-2b-Not1-Age1-R
3076	3003 H-4,5,6	Q 5	3076-myc-BsrG1-F + 3076-myc-Nhe1-Kpn1-R

Date:2/21/2018

#	Template	Restriction Enzymes	Note
1-5	3154 pMiniT2	EcoR I	wrong enzymes
7,8,10	3021 pMiniT2	EcoR I
11,14	3027 pMiniT2	EcoR I
Aim:	Test if we succeed to put insert into pMiniT 2

Digestion of 3154, 3021 or 3027 pMiniT2

Date:2/22/2018

#	products	Restriction Enzymes	Note
1,2	3010 L pMiniT2	EcoR I	1
3,4,5	3001 pMiniT2	EcoR I	4
6,7,8,9,10	3076 pMiniT2	EcoR I	7
11,12	3027 pMiniT2	EcoR I	11
Destrisprtion	We did miniprep and digested these products to test if we were sucessful.	1,4,7,11 to do sequencing

Date:2/23/2018

Digestion Test for 3154 Restriction enzymes:BamH I+ Kpn I

Digestion for 3154 from miniprep

Restriction Enzyme:Kpn I+ BamH I

Result: good, ordered for sequencing;sequencing result is good too.

Digestion of Vector 3154 for 3129 ligation Cut by Kpn I + Not I

We did ligation, but seems like the colonies are too much in a whole plate, so we planed to repeat but still picked up three colonies in this bad plate.

Digestion of Vetcor and Insert for 3129 ligation

Digestion of Vector and Insert for 3027 Ligation

Restriction Enzymes: Nhe I+ Not I

Because it looks like that the band of insert is not desired, so we repeated the digestion.

Another Digestion of Vector and Insert for 3027 Ligation

Restriction Enzymes:Nhe I+ Not I

We got the similar band of the old one.So just cut the very light color band out,not sure, so cut both two light bands.

Digestion of insert from 3076 pMiniT2  for 3076 ligation

Restriction Enzymes:BsrG I+ Kpn I

Not sure about the band, So planned to repeat next week.

PCR of 2493 to get the insert for 3021

2X PCR Mix:Platinum HiFi

Primers:3021-F2  + 3021-Age1-Xba1-R , Vector: BssHII - Nhe1

Good , so cut them and put into tube and add QG buffer