Cultivation: 30µLmother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.
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Synechococcus sp PCC 7942 had no signals of life.
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Week3(8/29/2016-9/4/2016)
pMV-G19
pMV-G15
Colonies were used to inoculate overnight cultures.
Plasmids were isolated using a miniprep kit.
Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli
19 amplification at 65.0°C with 19.rev/fwd primesPCR worked, positive control worked, no amplification of 19
15 amplification at 65.0°C with 15.rev/fwd primes PCR worked, positive control worked, no amplification of 15
A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
This result was confirmed by sequencing.
The fragments of 19 and 15 were purified with PCR Purification Kit.
Ligation of 15 with 19
Fragment of 19 was phosphorylated and then purified with DNA Purification Kit.
19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.A colony PCR was performed with five colonies
Gel electrophoresis showed that 5 colonies were positive for insertion of 19 .
Two of these colonies containing 19 were used to inoculate overnight cultures.
15 gene fragment was phosphorylated.Plasmids containing T vector with 19 (pT-19)were isolated using a miniprep kit.
Mono-restriction digest of pT-19 with stu I
The enzyme-digested product was dephosphorylation.
Dephosphorylated plasmid and phosphorylated gene 15 were connected.
Ligation product was transformed into E.coli via heat shock.
A colony PCR was performed with twelve colonies.
Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.
These two colonies were used to inoculate overnight cultures.
Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.
Mono-restriction digest of pT-19-15 with Nru I
The enzyme-digested product was dephosphorylation.
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火影忍者十分钟疾风传。了解其中最佳CP鸣樱 从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。
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火影忍者十分钟疾风传。了解其中最佳CP鸣樱 从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。
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终于来到喀纳斯——中国最西北的角落，此次西北之行可到达的最远处。在这个纬度高达 48 度的地方，山坡上的植被更加茂密；太阳下落得很慢，阳光以一个非常低的角度横扫过来，跨过群山，透过树木，在地上都投出长长的影子，非常好看。
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告别禾木乡 ，继续向喀纳斯方向前行，途中经过冲乎尔 乡。这里是一个天然牧场，坐落在小盆地，群山环抱，乡民主要以放牧为生。果然，不时就能看见牧羊人赶着几百头“阿勒泰 大尾羊”在公路上浩浩荡荡地走着，一路扬起滚滚烟尘，甚是壮观。
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一路北行，来到禾木 乡。这里几乎是中国的最西北角，与哈萨克斯坦、俄罗斯和蒙古等三国接壤。禾木地广人稀，和我去过的许多边境小城很像。穿行而过，不见几户人家，除了几个正在建设的小旅馆外，能零星看到的只是几个小木屋和蒙古包。
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继续在铁热克提 附近沿着蜿蜒的小道行摄，不知不觉间便绕到这座高山的另一侧。这里的杨树长得更高耸茂密，西斜的阳光恰到好处地将树梢照得金黄透亮，一道道树影投射在绒毛地毯般的甸上，构成近乎完美的光影，令人心旷神怡，不舍离。
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